JP2017538412A - Binding member for human C-MAF - Google Patents

Abstract

The present disclosure relates to binding members, particularly antibody molecules, that bind to human Maf. The binding member is useful for determining the expression level of Maf. The present invention provides, for example, a binding member or functional fragment or variant thereof that specifically binds to an epitope encoded by SEQ ID NO: 22, wherein the binding member or functional fragment or variant thereof is human c A heavy chain CDR1 that binds specifically to MAF and comprises the amino acid sequence of SEQ ID NO: 38, and / or a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and / or a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 42 And / or a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, and / or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and / or a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 30.

Description

Reference to the Sequence Listing The contents of the sequence listing ("3190_012PC01_SL. Txt", 77,454 bytes, prepared on December 4, 2015) filed with the present application and submitted electronically are hereby incorporated by reference in their entirety. In the book.

FIELD OF THE INVENTION The present invention relates to binding members, in particular antibody molecules, which bind to human c-MAF. Binding members are useful for quantifying c-MAF, diagnosis and prognosis of c-MAF related disorders, and treatment of c-MAF related disorders.

BACKGROUND Challenge Metastasis, a complex process caused by intricate interactions between tumor cells and surrounding normal tissue in a variety of important organs, accounts for 90 percent of all cancer deaths in patients with solid tumors. Occupy The molecular and cellular mechanisms that cause metastases to form in primary tumors must be understood in order to better cope with this major life-threatening problem. Identification of transgenes and mechanisms is essential for understanding the basic biology of this fatal condition and its relationship to clinical practice. Previous studies have led to the recognition that the metastatic process is complex, but how and why metastasis occurs, and what mechanisms make it a tissue-specific process, the nuclear power plant Many years after excision of a sexual tumor, what kind of event makes the quiescent metastasis active and dead, what kind of metastasis-mediated gene will ultimately be a valuable diagnostic marker and treatment It was not possible to explain whether it became a target.

  The present invention provides that the reliable identification of markers that predict bone metastasis provides a preventative therapeutic opportunity by imposing limitations on the spread and metastatic growth of bone metastatic tissue by cancer cells. It is based on the recognition of delaying or altering the lethal condition as well as the need for a mechanism to identify the expression of metastasis predictive markers. Thus, for example, it has been shown that the accumulation of proteins and mRNA of MAF (true breast cancer bone transposon) can be obtained by 16q22-24 (16q23) amplification or 16q23 translocation, among other potential mechanisms. c-MAF is also responsible for driving cancer bone metastatic lesions, including osteolytic cancer bone metastases.

  In one embodiment, the present invention relates to the detection of genetic abnormalities by the use of antibodies, as well as to the prognosis and / or diagnosis of metastasis (eg bone metastasis) in cancer based thereon. In one embodiment, the invention comprises the use of an antibody to determine the level of a gene of interest in a primary tumor sample. In one embodiment, the invention relates to a binding member (eg, an antibody) that specifically binds human c-MAF. Similarly, the invention also relates to a method for designing a personalized treatment in a subject having cancer, comprising using an antibody to determine the level of a gene of interest in a sample. In one embodiment, the gene of interest is MAF. In another embodiment, the cancer is breast cancer, lung cancer, prostate cancer, or kidney cancer.

  In one aspect, the invention is directed to a binding member or functional fragment or variant thereof that specifically binds to the epitope encoded by SEQ ID NO: 22. In some embodiments, the binding member or functional fragment or variant thereof specifically binds human c-MAF, wherein the binding member or functional fragment or variant thereof comprises the amino acid sequence of SEQ ID NO: 38 Heavy chain CDR1 comprising and / or heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and / or heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 42; and / or light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, And / or light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and / or light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 30.

  In some embodiments, the binding member is an antibody. In some embodiments, the antibody is a rabbit antibody, a chimeric antibody or a humanized antibody.

  In some embodiments, the binding member or functional fragment or variant thereof comprises a VH domain having a sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, a binding member or functional fragment or variant thereof comprises a VH domain having a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, a binding member or functional fragment or variant thereof comprises a VH domain having a sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the binding member or (of) a functional fragment or variant thereof comprises a VH domain having a sequence comprising the amino acid sequence of SEQ ID NO: 17.

  In some embodiments, a binding member or functional fragment or variant thereof comprises a VL domain having a sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, a binding member or functional fragment or variant thereof comprises a VL domain having a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, a binding member or functional fragment or variant thereof comprises a VL domain having a sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 21. In some embodiments, the binding member or functional fragment or variant thereof comprises a VL domain having a sequence comprising the amino acid sequence of SEQ ID NO: 21.

  In some embodiments, a binding member or functional fragment or variant thereof comprises a heavy chain sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, a binding member or functional fragment or variant thereof comprises a heavy chain sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, a binding member or functional fragment or variant thereof comprises a heavy chain sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, a binding member or functional fragment or variant thereof comprises a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 16.

  In some embodiments, a binding member or functional fragment or variant thereof comprises a light chain sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, a binding member or functional fragment or variant thereof comprises a light chain sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 20.

  19. The binding member or functional fragment or variant thereof according to claim 18, comprising a light chain sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody or fragment thereof comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 20.

  In some embodiments, the invention is directed to a polynucleotide encoding any binding member described herein or a functional fragment or variant thereof. In some embodiments, the polynucleotide encodes a polypeptide encoding an antigen binding molecule or a fragment thereof. In some embodiments, the binding member or functional fragment or variant thereof is an antibody.

  In some embodiments, the polynucleotide comprises a VH domain having a sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the polynucleotide encodes a VH domain having a sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the polynucleotide encodes a VH domain having a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 15. In some embodiments, the polynucleotide encodes a VH domain having a sequence identical to the nucleotide sequence of SEQ ID NO: 15.

  In some embodiments, the polynucleotide encodes a VL domain having a sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encodes a VL domain having a sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encodes a VL domain having a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 20. In some embodiments, the polynucleotide encodes a VL domain having a sequence identical to the nucleotide sequence of SEQ ID NO: 20.

  In some embodiments, the polynucleotide encodes a heavy chain having a sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the polynucleotide encodes a heavy chain having a sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the polynucleotide encodes a heavy chain having a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 14. In some embodiments, the polynucleotide encodes a heavy chain having a sequence identical to the nucleotide sequence of SEQ ID NO: 14.

  In some embodiments, the polynucleotide encodes a light chain having a sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide encodes a light chain having a sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide encodes a light chain having a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide encodes a light chain having a sequence identical to the nucleotide sequence of SEQ ID NO: 18.

  In some embodiments, the amino acid encoded by the polynucleotide binds to the epitope set forth in SEQ ID NO: 22.

  In some embodiments, a binding member or functional fragment or variant thereof binds human c-MAF with an affinity (KD) of at least about 1.5 nM or less. In some embodiments, a binding member or variant thereof binds human c-MAF with an affinity (KD) of at least about 1.2 nM or less. In some embodiments, a binding member or variant thereof binds human c-MAF with an affinity (KD) of at least about 1.1 nM or less.

  In some embodiments, the present invention is directed to a vector comprising any of the polynucleotides disclosed herein.

  In some embodiments, the present invention comprises any of the polynucleotides disclosed herein or any of the vectors disclosed herein or any of the disclosed herein Host cells expressing binding members are of interest. In some embodiments, the present invention is directed to a method of producing an antigen binding member comprising culturing a host cell. In some embodiments, the present invention is directed to methods using antigen binding members produced by host cells or said methods or production for detecting c-MAF.

  In some embodiments, the present invention is directed to binding members or functional fragments or variants thereof that compete with INB-1-11-8 for binding to the epitope encoded by SEQ ID NO: 22.

FIG. 1 shows a comparison of the two isoforms of c-MAF (short isoform (MAF (short)) and long isoform (MAF (long)).

FIG. sapiens, P .; troglodytes, B. Taurus, M. musculus, R., et al. norvegicus, G. gallus, and D. Figure 7 shows a comparison of the amino acid sequences of c-MAF from rerio.

A) Antigen-specific binding results of Elisa. INB-1-11-8 was tested at a range of dilutions to assess its specificity for antigen binding. As a result, it was confirmed that antigen affinity was maintained even at dilutions of more than 1: 500. B) The specificity of INB-1-11-8 antibody was tested by Western blot. The antibody was used at 1:50 dilution using 3% BSA as blocking solution. The antibody specifically recognized the endogenous c-MAF isoform (including post-translationally modified proteins) and its degraded form. Also, it specifically recognized the recombinantly expressed long and short human c-MAF and mouse c-MAF isoforms. MCF7 and T47 are ER + breast cancer cell lines. BoM2 is a bone metastatic MCF7 derivative. 293 T cells are kidney cells C) Representative c-MAF immunohistochemical image of primary breast cancer tissue. Images in the left column represent c-MAF positive tumors. Right column is c-MAF negative tumors. The above image was stained with INB-1-11-8 antibody against Maf. The lower image (taken using the Santa Cruz Biotechnology Inc. M153 antibody) is provided for comparison.

FIG. 4 shows SDS-PAGE analysis of commercial BSA and cMaf (Q1) samples.

FIG. 5 shows an exemplary sensorgram of CFCA analysis of cMaf (Q1).

FIG. 6 shows kinetic analysis of c-MAF (Q1) binding to INB-1-11-8.

FIG. 7 shows a single concentration fit of c-MAF (Q1) binding to INB-1-11-8.

FIG. 8 shows the heavy and light chain sequences of c-MAF antibody INB-1-11-8.

A) IHC score. Model evaluation: histogram plot. B) Plot of sensitivity and specificity. C) Receive operating curve (ROC).

FIG. 10 shows baseline characteristic bivariate analysis.

A) Kaplan-Meier curve showing the probability of boneless metastasis. B) Plot of cumulative bone metastasis incidence.

Detailed Description of the Invention Definitions of General Terms and Expressions As used herein, “and / or” refers to each of the specified features or components with or without the other. It should be considered as a specific disclosure. For example, “A and / or B” may specifically disclose each of (i) A, (ii) B and (iii) A and B as if each was separately described herein. Should be regarded as

  As used herein, "binding member" refers to one member of a set of molecules that bind one another. The members of the binding pair may be of natural origin or may be wholly or partially synthetically produced. One member of one set of molecules binds to the other member of one set of molecules and thus has a surface area or cavity complementary to the particular spatial and polar configuration of the other member. Examples of types of binding pairs are antigen-antibody, receptor-ligand and enzyme-substrate. In some embodiments, the binding member is an antibody. In some embodiments, the binding member is an antibody that binds to c-MAF antigen.

  As used herein, "CDR regions" or "CDRs" are described by Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Edition. It is intended to show the hypervariable regions of heavy and light chains of immunoglobulins as defined by the US Department of Health and Human Services, Public Service, NIH, Washington. The antibody typically contains three heavy chain CDRs designated HCDR1, HCDR2 and HCDR3 and three light chain CDRs designated LCDR1, LCDR2 and LCDR3. The term CDR (s) refers to one or more of these regions that contain most of the amino acid residues involved in binding, depending on the affinity of the antibody for the antigen or epitope that it recognizes. Or as used herein to indicate the whole. Among the six CDR sequences, the third CDR of the heavy chain (HCDR3) is known in the art as the V (D) J rearrangement of the V, D and J gene segments of the germline immunoglobulin heavy chain locus. Essentially due to known mechanisms, it has the greatest degree of variability (ie, greater diversity). HCDR3 may be as short as 2 amino acids, or as long as 26 amino acids, or may have any length between these two extremes. The length of the CDRs may also vary according to the length that can be accommodated by the particular underlying framework. Functionally, HCDR3 may play an important role in determining antibody specificity (Segal et al. (1974) Proc Natl Acad Sci USA. 71 (11): 4298-302; Amit et al. (1986) Science 233 (4765): 747-53; Chothia et al., (1987) J. Mol. Biol. 196 (4): 901-17; Chothia et al., (1989) Nature 342 (6252): 877-83; 1990) J. Immunol. 144 (5): 1965-8; Sharon (1990a) PNAS USA. 87 (12): 4814-7, Sharon (1990b) J. Immunol. 144: 4863-4869, Kabat et al. ) Sequen es of Proteins of Immunological Interest, 5th Edition.US Department of Health and Human Services, Public Service, NIH, Washington).

As used herein, an "antibody", "antibody molecule" or "antibodies" may be naturally produced or partially or wholly synthetically produced. Regardless, it represents an immunoglobulin. The term also encompasses any polypeptide or protein comprising an antibody antigen-binding site. The present invention does not relate to naturally occurring forms of the antibodies (in other words, they are not in their natural environment, but they can be isolated or obtained by purification from natural sources or by genetic recombination It should be understood herein that they can be obtained by chemical synthesis or, and thus, they can contain non-naturally occurring amino acids. Antibody fragments that contain the antigen binding site of the antibody include, but are not limited to, molecules such as Fab, Fab ′, F (ab ′) ₂ , Fab′-SH, scFv, Fv, dAb, and Fd. For example, various other antibody molecules comprising one or more antibody antigen binding sites are engineered, including, for example, Fab2, Fab3, diabodies, triabodies, tetrabodies, camelbodies, nanobodies and minibodies. ing. Antibody molecules and methods for their construction and use are described in Hollinger & Hudson (2005) Nature Biot. 23 (9): 1126-1136.

  As used herein, "antibody molecule" should be construed to encompass any binding member or substance having an antigen binding site of an antibody with the necessary specificity and / or binding to an antigen. is there. Thus, the term encompasses functional antibody fragments and derivatives comprising any polypeptide comprising the antigen binding site of the antibody, whether natural or wholly or partially synthetic. Thus, included are chimeric molecules or equivalents comprising the antigen binding site of an antibody (eg, derived from another species or belonging to another antibody class or subclass) fused to an antibody. The cloning and expression of chimeric antibodies are for example described in European Patent Application Publication No. 0120694A (Boss et al.) And European Patent Application Publication No. 0125023A (Cabilly et al.), Which are incorporated herein by reference in their entirety. )It is described in.

  As used herein, for example, a "functional fragment or variant" of a binding member of the invention is capable of specifically binding at least some functions of the complete binding member (e.g., an antigen such as Maf) Means a fragment or variant of a binding member that holds

  As used herein, the term "amplification of a gene" refers to the process by which various copies of a gene or gene fragment are formed in individual cells or cell lines. The copies of the gene are not necessarily located on the same chromosome. The overlapping region is often referred to as the "amplicon". Usually, the amount of mRNA produced, ie, the gene expression level, increases in proportion to the copy number of a particular gene.

  As used herein, “MAF gene”, “Maf”, “c-MAF” or “c-Maf” (v-Maf myodenoid fibrosarcoma oncogene homolog also known as Maf or MGC71685 ( Avians) are transcription factors that include leucine zippers that act like homodimers or heterodimers. Depending on the DNA binding site, the encoded protein may be a transcriptional activator or transcriptional repressor. The DNA sequence encoding MAF is listed in the NCBI database as accession number NG_016440 (SEQ ID NO: 1) (code). The genomic sequence of MAF is shown in SEQ ID NO: 13. The methods of the invention may utilize either coding sequences or genomic DNA sequences. From the above DNA sequences, two messenger RNAs are transcribed, each of which gives rise to one of two c-MAF protein isoforms, alpha isoform and beta isoform. The complementary DNA sequences for each of the above isoforms are listed in the NCBI database as accession numbers NM_005360.4 (SEQ ID NO: 2) and NM — 001031804.2 (SEQ ID NO: 3), respectively. Additional information regarding isoforms of c-MAF can be found in Eychene et al., NRC 8: 683-693 (2008), which is incorporated herein by reference in its entirety. In some embodiments, the present invention relates generally to cancer (eg, International Patent Application No. PCT / IB2013 / 001204 and International Patent Application No. PCT / ES2011 / 070693 and US Patent Application No. 13 / 878,114). And US patent application Ser. No. 13 / 878,114 (triple negative breast cancer and ER + breast cancer), International patent application PCT / US2014 / 026154 (renal cell carcinoma), International patent application PCT / US2014 / 028722 (breast cancer), International Patent Application No. PCT / US2013 / 044584 (lung cancer), US Patent Application No. 14 / 050,262 and International Patent Application No. PCT / IB2013 / 002866 (prostate cancer), International Patent Application No. PCT / US2014 / 059506 Issue (HER 2 + breast cancer) Prognostication of US Patent Application No. 14 / 213,670 and International Patent Application No. PCT / US2014 / 028569 (metastatic cancer) (each is incorporated herein by reference in its entirety) The use of the c-MAF gene for

  In the context of the present invention, “functionally equivalent variants of c-MAF protein” are (i) conserved or non-conserved amino acid residues of one or more of the amino acid residues (preferably, conserved) Variant of the c-MAF protein (SEQ ID NO: 4 or SEQ ID NO: 5), wherein such substituted amino acid residues are the amino acid residues encoded by the genetic code, substituted by May or may not) or (ii) contains an insertion or deletion of one or more amino acids and has the same function as the c-MAF protein, ie acts as a DNA binding transcription factor Is understood as a variant to Variants of the c-MAF protein are the ability of c-MAF to promote cell growth in vitro, as set out in International Patent Application Publication WO 2005/046731, which is hereby incorporated by reference in its entirety. Cyclin D2 promoter or c-MAF responsive region (MARE or in the cells expressing c-MAF as described in WO2008 / 098351, which is incorporated herein by reference in its entirety) The ability of an inhibitor or test compound to block the transcriptional ability of a reporter gene under the control of a promoter containing a c-MAF response element) can be identified using methods. Variants of c-MAF may also be stimulated by PMA / ionomycin in cells expressing NFATc2 and c-MAF as described in US2009 / 048117A, which is incorporated herein by reference in its entirety. It can be identified using methods based on the ability of so-called inhibitors to block expression of the reporter gene under control of the IL-4 promoter in response.

  The c-MAF variants according to the invention are preferably at least about 50%, at least about 60%, at least about 70%, preferably with the amino acid sequence of any c-MAF protein isoform (SEQ ID NO: 4 or SEQ ID NO: 5). At least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or Have at least about 99% sequence similarity. The degree of similarity between the variant and the particular c-MAF protein sequence defined above is determined using algorithms and computer processes widely known to those skilled in the art. The similarity between two amino acid sequences is preferably determined according to the BLASTP algorithm [BLAST Manual, Altschul, S. et al. , NCBI NLM NIH Bethesda, Md. 20894, Altschul, S.A. Et al. Mol. Biol. 215: 403-410 (1990)].

  As used herein, the mammalian target of rapamycin (mTOR) or "mTor" refers to the protein corresponding to EC 2.7.11.1. The mTor enzyme is a serine / threonine protein kinase that regulates cell growth, cell motility, cell growth, cell survival and transcription.

  As used herein, an "mTor inhibitor" is capable of completely or partially inhibiting the expression of the mTor gene (preventing the production of the expression product of the above gene (transfer of the mTor gene Refers to any molecule) both by blocking and / or blocking the translation of mRNA derived from mTor gene expression) and by directly inhibiting mTor protein activity. Including dual or more targets and also inhibitors with mTor protein activity among them.

  As used herein, "Src" refers to a protein corresponding to EC 2.7.10.2. Src is a non-receptor tyrosine kinase and proto-oncogene. Src may play a role in cell growth and embryonic development.

  As used herein, “Src inhibitor” is capable of completely or partially inhibiting the expression of Src gene (preventing the production of the expression product of the above gene (transcription of Src gene Refers to any molecule) both by blocking and / or blocking the translation of mRNA derived from Src gene expression) and by directly inhibiting Src protein activity.

  As used herein, “prostaglandin-endoperoxide synthase 2”, “cyclooxygenase-2” or “COX-2” refers to a protein corresponding to EC 1.14.99.1. Point to. COX-2 is involved in the conversion of arachidonic acid to prostaglandin endoperoxide H2.

  As used herein, “COX-2 inhibitor” can completely or partially inhibit the expression of the COX-2 gene (preventing the production of the expression product of the above gene (COX -2) any molecule (both by blocking the transcription of the gene and / or blocking the translation of mRNA derived from COX-2 gene expression) and by directly inhibiting COX-2 protein activity .

  As used herein, "outcome" or "clinical outcome" refers to the resulting disease course and / or disease progression, eg, relapse, time to relapse, metastasis, metastasis The period of time may be characterized by the number of metastases, the number of sites of metastases and / or death due to the disease. For example, good clinical outcomes include cure, prevention of recurrence, prevention of metastasis and / or survival within a period of time (no recurrence), and poor clinical outcomes include disease progression, metastasis and / or There is death within a certain period.

  As used herein, the term "expression level" of a gene refers to a measurable amount of gene product produced by the gene in a sample of a subject, wherein the gene product is , Transcript or translation product. Thus, expression levels can relate to nucleic acid gene products (eg, mRNA or cDNA) or polypeptide gene products. The expression level is obtained from the sample of the subject and / or the reference sample (s) and may, for example, be detected de novo or correspond to previous determinations. Expression levels may be determined or measured, for example, using microarray methods, PCR methods (eg, qPCR) and / or antibody-based methods, as known to those skilled in the art. In some embodiments, expression levels of c-MAF are measured using the antibodies disclosed herein.

  As used herein, the term "gene copy number" refers to the copy number of a nucleic acid molecule in a cell. Gene copy number includes the gene copy number in the genomic (chromosomal) DNA of a cell. In normal cells (non-tumor cells), the gene copy number is usually 2 copies (1 copy in each member of the chromosome pair). The gene copy number sometimes includes half of the gene copy number taken from a sample of the cell population.

  "Elevated expression level" is understood as the expression level when it refers to the level of the MAF gene above the level of the MAF gene in the reference or control sample. Elevated levels can be triggered by amplification or translocation of one gene or the 16q23 or 16q22-24 chromosomal locus without excluding other mechanisms. In particular, the expression level in the sample isolated from the patient is at least about 1.1, about 1.5, about 5, about 10, about 20, about 30 as compared to the reference or control. A sample is considered to have high c-MAF expression levels when about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 90 times, about 100 times or even higher Can be

  "Probe," as used herein, refers to an oligonucleotide sequence that is complementary to a specific nucleic acid sequence of interest. In some embodiments, the probe may be specific to a region of the chromosome known to cause translocation. In some embodiments, the probe has a specific label or tag. In some embodiments, the tag is a fluorophore. In some embodiments, the probe is a DNA in situ hybridization probe whose label is based on stable coordinate binding of platinum to nucleic acids and proteins. In some embodiments, the probes are described in US patent application Ser. Nos. 12 / 067,532 and 12 / 181,399, which are incorporated by reference in their entirety, or Swennenhuis et al., "Construction of repeat-free fluorescence in situ hybridization probes", Nucleic Acids Research 40 (3): e20 (2012).

  "Tag" or "label" as used herein directly or indirectly with the probe, which allows to visualize, mark or otherwise capture the position of the probe or probe Refers to any physical molecule that is associated.

  "Translocation", as used herein, refers to exchanging chromosomal material between chromosomes in unequal or equal amounts. In some cases, translocations occur on the same chromosome. In some cases, translocations occur between different chromosomes. Translocations occur frequently in many types of cancer, including breast cancer and leukemia. The translocation may be a primary reciprocal translocation or a more complex secondary translocation. There are several primary translocations involving the immunoglobulin heavy chain (IgH) locus, which are thought to constitute many cancer initiating events (Eychene, A., Rocques, N., and Puoponnot, C 2008. Nature Reviews: Cancer. 8: 683-693) .. A New MA Fia in cancer.

  "Ploidy" or "ploidy" as used herein indicates that the cell contains more than two copies of the gene of interest. In some cases, the gene of interest is MAF. In some embodiments, polyploidy is associated with accumulation of expression of a gene of interest. In some embodiments, ploidy is associated with genomic instability. In some embodiments, genomic instability can lead to chromosomal translocations.

  "Whole genome sequencing" as used herein is a process in which the entire genome of an organism is sequenced in one go. For example, Ng. , P .; C. and Kirkness, E.S. F. , Whole Genome Sequencing. 2010. Methods in Molecular Biology. 628: 215-226.

  "Exome sequencing" as used herein is a process by which the entire coding region of an organism's DNA is sequenced. In exome sequencing, mRNA is sequenced. Untranslated regions of the genome are not included in exome sequencing. For example, Choi, M et al., Genetic diagnosis by whole exome capture and massively parallel DNA sequencing. 2009. PNAS. 106 (45): 19096-19101.

  "Metastasis" as used herein is understood as the spread of cancer from the organ where the cancer first originated to a different organ. Metastasis usually occurs through the blood or lymphatic system. When cancer cells spread and form new tumors, the latter are called secondary or metastatic tumors. Cancer cells forming secondary tumors are similar to those of the original tumor. If breast cancer, for example, spreads to the lungs (metastasizes), secondary tumors are formed from malignant breast cancer cells. The disease in the lung is metastatic breast cancer, not lung cancer.

  "Predicting" as used herein refers to the determination of the likelihood that a subject suffering from cancer will metastasize to a distant organ. As used herein, "good prognosis" is expected to be at low risk for the subject to survive or / or have no recurrence or distant metastasis within a certain period of time (e.g. , To be predicted). The term "low" is a relative term and in the context of the present application refers to the risk of the "low" expression group with respect to clinical outcome (relapse, distant metastasis etc). A "low" risk may be considered as a lower risk than the average risk for a heterogeneous cancer patient population. In the study of Paik et al. (2004), the overall "low" risk of recurrence was considered to be less than 15 percent. The risk also fluctuates depending on the period. The period may be, for example, five, ten, fifteen or even twenty years after the initial diagnosis of cancer or after a prognosis has been made.

  As used herein, "poor prognostic" is predicted to be at high risk for the subject not having survival and / or having or having recurrence or distant metastasis within a fixed period of time, eg, Indicates that it is expected. The term "high" is a relative term and in the context of the present application refers to the risk of the "high" expression group with respect to clinical outcome (relapse, distant metastasis etc). A "high" risk may be considered as a risk higher than the average risk for heterogeneous cancer patient populations. In the study of Paik et al. (2004), the overall "high" risk of recurrence was considered to be greater than 15 percent. The risk also fluctuates depending on the period. The period of time may be, for example, five, ten, fifteen or even twenty years after the initial diagnosis or prognosis of the cancer has been made.

  "Reference value" as used herein refers to a test value used as a reference to a value / data obtained by laboratory test of a patient or a sample collected from the patient. Reference values or reference levels are absolute values; relative values; values with upper and / or lower limits; series of values; average value; median value, mean value, or a specific control value or base It may be a value to be compared to the line value. Reference values may be based on individual sample values, such as, for example, values obtained from a sample from a subject being tested, but may be based on values at earlier time points. The reference value may be based on a large number of samples (e.g., from a group of subjects of a calendar age-matching group) or may be based on a pool of samples that includes or excludes the sample to be tested.

  As used herein, "subject" or "patient" refers to all animals classified as mammals, including domestic and farm animals, primates and humans, such as Including, but not limited to, humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats or rodents. Preferably, the subject is a human male or female of any age or race.

  The term "treatment" as used herein, terminates, prevents, ameliorates, or enters a clinical condition as described herein. Refers to any type of treatment that aims to reduce morbidity. In a preferred embodiment, the term treatment relates to the prophylactic treatment of a disorder or condition as defined herein (ie, a treatment that reduces the susceptibility to a clinical condition). Thus, the terms "treatment", "treat" and their equivalents refer to the desired pharmacological or physiological effects, including any treatment of a pathological condition or disorder in mammals, including humans. Point to get. The effect may be prophylactic with regard to preventing the disorder or its symptoms completely or partially, and / or therapeutic with respect to partial or complete cure for the disorder and / or adverse effects that may result from the disorder. It can be. That is, “treatment” includes (1) preventing the occurrence of a disorder or recurrence in a subject, (2) inhibiting the disorder, eg, arresting its development, (3) eg, losing By reconstituting or repairing a defective, defective or defective function or stimulating an inefficient process, so that the host is no longer afflicted with the disorder or its symptoms, the disorder or related thereto Stopping or terminating at least one symptom (eg causing a disorder or regression of the symptom), or (4) reducing, alleviating or ameliorating the disorder or symptoms associated therewith (herein Recovery is at least the magnitude of the parameter (eg, inflammation, pain or immunodeficiency) Including a wide use in the sense) to refer to a decrease. In some embodiments, the treatment is to prevent bone degradation. In some embodiments, the treatment comprises International Patent Application No. PCT / IB2013 / 001204, US Provisional Patent Application No. 61 / 801,769, US Provisional Patent Application No. 61 / 801,642, US Provisional Patent Application No. 61 / 801,718, International Patent Application No. PCT / US2013 / 045458, US Provisional Patent Application No. 61 / 713,318 and International Patent Application No. PCT / US2014 / 059506 (incorporated herein by reference in its entirety) Is any treatment disclosed or discussed in

  As used herein, "sample" or "biological sample" refers to biological material isolated from a subject. The biological sample may comprise any biological material suitable for determining the expression level of c-MAF gene. The sample may be isolated from any suitable biological tissue or body fluid such as, for example, tumor tissue, blood, plasma, serum, urine or cerebrospinal fluid (CSF).

  A "tumor tissue sample" is understood as a tissue sample originating from a primary cancer tumor. The above samples can be obtained by conventional methods, for example by biopsy using methods well known to the person skilled in the relevant medical technology.

  "Erosive bone metastasis" refers to the type of metastasis in which bone resorption (progressive loss of bone density) caused by stimulation of osteoclast activity by tumor cells refers to the type of metastasis that occurs in the vicinity of metastasis. It is characterized by pain, pathological fractures, hypercalcemia, spinal cord compression, and other syndromes resulting from nerve compression.

Binding Member Binding member usually comprises a molecule having a binding site. For example, the binding member may be an antibody molecule or non-antibody protein comprising a binding site. The binding site may be obtained by placing the CDRs on antibody framework regions and / or on non-antibody protein scaffolds (such as fibronectin or cytochrome B) (Haan & Maggos (2004) BioCentury, 12 (5): A1- A6; Koide et al. (1998) J. Mol. Biol. 284: 1141-1151; Nygren et al. (1997) Curr. Op. Struct. Biol. 7: 463-469), or the amino acid residue of the loop within the protein scaffold. The groups can be provided by randomizing or mutating them to impart binding specificity to the desired target. Scaffolds for engineering novel binding sites in proteins are reviewed in detail by Nygren et al., Supra. A protein scaffold for antibody mimics is disclosed in International Patent Application Publication No. WO 00/034784 A1 (Lipovsek), among which the present inventors have found that fibronectin type III domain having at least one randomized loop And proteins (antibody mimics) are described. Suitable scaffolds for grafting one or more CDRs (eg, a set of HCDRs) can be provided by any domain member of the immunoglobulin gene superfamily. The scaffold may be human or non-human protein. An advantage of a non-antibody protein scaffold is that it can provide an antigen binding site in a scaffold molecule that is smaller and / or easier to manufacture than at least some antibody molecules. Small size binding members have useful physiological properties, such as the ability to invade cells, penetrate deeply into tissues, or reach targets in other structures, or bind to protein antigens within the target antigen. Can be given. The use of antigen binding sites in non-antibody protein scaffolds is reviewed by Wess, (2004) In: BioCentury, The Bernstein Report on BioBusiness, 12 (42), A1-A7. Typical are proteins having a stable backbone and one or more variable loops, wherein the amino acid sequence of one or more loops to create an antigen binding site that binds to a target Protein (s) is a protein that has been specifically or randomly mutated. Examples of such proteins include S. Included are the IgG binding domain of protein A from S. aureus, transferrin, tetranectin, fibronectin, lipocalin and gamma crystals and other AffilinTM scaffolds (Scil Proteins).

  Other approaches include synthetic "microbodies" based on cyclotides, small proteins with intramolecular disulfide bonds, microproteins (VersabodiesTM, Amunix) and ankyrin repeat proteins (DARPins, Molecular Partners) Can be mentioned.

  In addition to antibody sequences and / or antigen binding sites, binding members according to the invention may, for example, form a peptide or polypeptide (e.g. a folded domain) or otherwise function in addition to the ability to bind an antigen. Other amino acids may be included that impart properties to the molecule. Binding members of the invention may carry a detectable label or may be conjugated to a toxin or targeting moiety or enzyme (eg, via a peptidyl bond or linker). For example, the binding member may comprise a catalytic site (eg, in an enzyme domain) and an antigen binding site, wherein the antigen binding site binds to the antigen and directs the catalytic site to the antigen. The catalytic site may inhibit the biological function of the antigen, for example by cleavage.

In some embodiments, the binding member is an antibody. As mentioned above, while the CDRs of an antibody may be carried by a non-antibody scaffold, the structure for carrying the CDR or set of CDRs of the present invention is an immunoglobulin gene in which the CDR or set of CDRs has been rearranged An antibody heavy chain sequence or antibody light chain sequence or a substantial portion thereof located at a position corresponding to the CDR or set of CDRs of the naturally occurring V _H and V _L antibody variable domains encoded by There are many. The structure and location of immunoglobulin variable domains can be determined by reference to Kabat et al. (1991) (supra) and modified versions thereof. Several academic and commercial online resources are available to query this database. See, for example, Martin, (1996) PROTEINS: Structure, Function and Genetics, 25: 130-133 and related online resources (current web address https://www.bioinf.org.uk/abs/simkab.html). Of.

  Utilizing monoclonal and other antibodies, it is possible to produce other antibodies or chimeric molecules that bind the target antigen using techniques of recombinant DNA technology. Such techniques may involve introducing the DNA encoding the immunoglobulin variable region or CDRs of the antibody into the constant region of a different immunoglobulin, or into the constant region and the framework region. See, for example, European Patent Application Publication No. EP 0184187A (Kudo et al.) Or European Patent Application Publication No. EP 0 239 400 A (Winter). The hybridoma or other cell producing the antibody may be subjected to genetic mutations or other changes, which may or may not alter the binding specificity of the antibody produced.

  Additional techniques available in the art of antibody engineering have made it possible to isolate human and humanized antibodies. For example, human hybridomas can be made as described in Kontermann & Dubel (2001) Antibody Engineering, Springer-Verlag New York, LLC; ISBN: 3540413545. Phage display (another established technique for generating binding members) is described in detail in many publications such as Kontermann & Dubel (above) and International Patent Application Publication WO 92/01047 A1 (McCafferty et al.) .

  Transgenic mice in which the mouse antibody gene has been inactivated and functionally replaced with human antibody genes, leaving intact alternative components of the mouse immune system can be used to isolate human antibodies. (Mendez et al. (1997) Nature Genet. 15 (2): 146-156). Alternatively, the method described by Grawunder & Melchers (International Patent Publication WO 03/068819 A1) can be used to generate genetically modified vertebrate precursor lymphocytes for the production of heterologous antibodies or binding proteins. In some embodiments, rabbits are used to generate antibodies. In some embodiments, antibodies are produced using hybridoma supernatants, recombinant antibody transient expression methods, or recombinant antibody stable cell line development and production methods. In some embodiments, the antibody is optionally purified using protein A. Humanized antibodies can be generated using techniques known in the art, such as, for example, those disclosed in International Patent Application Publication WO 91/09967 A1 (Adair et al.). In addition, International Patent Application Publication WO 04/006955 A1 (Foote) provides standard CDR structural types for CDR sequences of non-human antibody variable regions, live human antibody sequences (eg, germline antibody gene segments) Methods have been described for humanizing antibodies based on selecting variable region framework sequences from human antibody genes by comparison to standard CDR structural forms of the corresponding CDRs from Larry. Human antibody variable regions having standard CDR structural types similar to non-human CDRs form a subset of member human antibody sequences for selecting human framework sequences. Subset members can be further ranked by amino acid similarity between human and non-human CDR sequences. In the method of WO 04/006955 A1 (described above), human frameworks of selected subset members are used to construct a chimeric antibody in which human CDR sequences are functionally replaced by non-human CDR counterparts. To provide a framework sequence for providing high affinity and low immunogenicity humanized antibodies without the need for comparison of framework sequences between non-human and human antibodies. , Top ranked human sequences are selected. Also disclosed is a chimeric antibody produced according to the above method.

It has been shown that fragments of whole antibodies can perform the function of binding to an antigen. Examples of binding fragments are: (i) Fab fragments consisting of V _L , V _H , C _L and C _H 1 domains; (ii) Fd fragments consisting of V _H and C _H 1 domains; (iii) single antibodies Fv fragment consisting of V _L and V _H domains; (iv) dAb fragment consisting of V _H or V _L domains (Ward et al. (1989) Nature 341 (6242): 544-6; McCafferty et al. (1990) Nature 348 (6301): 552-4; Holt et al. (2003) Trends in Biotechnology 21: 484-490); (v) isolated CDR regions; (vi) F (ab ') ₂ fragments (two linked. Bivalent fragments containing Fab fragments), (Vii) Single-chain Fv molecules (scFv) (V _{) which} are linked V _H and V _L domains by a peptide linker that allows the two domains to associate to form an antigen binding site (Bird et al. 1998) Science 242 (4877): 423-6; Huston et al. (1988) PNAS USA, 85: 5879-5883); (viii) bispecific single chain Fv dimer (International Patent Application Publication WO 93 / No. 011161 Al (Whitlow et al.), As well as (ix) "diabodies" (multivalent or multispecific fragments constructed by gene fusion) (Holliger et al., 1993) PNAS USA. 90 (14): 6444-8 and International Patent Application Publication No. WO 94/13804 A1). Fv, scFv, or diabody molecules can be stabilized by the incorporation of a disulfide bridge linking the V _H and V _L domains (Reiter et al. (1996) Nature Biotech, 14: 1239-1245). Minibodies comprising a scFv is coupled to the C _H 3 domain may also be made (Hu et al., (1996) Cancer Res.56: 3055-3061 ). Another example of a binding fragment is the Fab 'by the addition of a small number of residues (containing one or more cysteines from the antibody hinge region) to the carboxyl terminus of the heavy chain C _H 1 domain. And Fab'-SH, which are Fab 'fragments in which the cysteine residue (s) of the constant domain have a free thiol group. An antibody molecule containing only two CDRs linked by a framework region has also been described (Qui et al. (2007) Nat. Biotechnol. 25: 921-929). CDR3 derived from the V _H domain or V _L domain was linked to the CDR1 or CDR2 loop of the other domain by ligation from the C-terminus of the selected CDR1 or CDR2 to the N-terminus of CDR3 via the FR region.

Domain antibodies (dAbs) are small monomeric antigen binding fragments of antibodies (i.e. variable regions of antibody heavy or antibody light chains) (Holt et al. (2003) Trends in Biotechnology 21: 484-490). V _H dAbs occur naturally in camelids (eg, camels, llamas), immunize them with target antigens, isolate antigen-specific B cells, and derive individual B cell-derived dAb genes It can be produced by direct cloning; however, dAbs can also be produced in cell culture. Binding members of the invention are dAbs comprising a V _H or V _L domain substantially as set forth herein, or a V _H or V _L domain substantially comprising the set of CDRs set forth herein. possible.

  The antibody fragments of the invention may be obtained from any of the antibodies disclosed herein by methods such as digestion with enzymes such as pepsin or papain and / or by cleavage of disulfide bridges by chemical reduction. In another method, antibody fragments encompassed by the present invention may be obtained by genetic recombination techniques well known to those skilled in the art, otherwise by peptide synthesis or by nucleic acid synthesis and expression.

  Functional antibody fragments according to the invention include any functional fragments whose half-life has been increased by chemical modification, in particular by pegylation, or by, for example, incorporation in liposomes.

  Bispecific or bifunctional antibodies form a second generation of monoclonal antibodies in which two different variable regions are combined in the same molecule (Holliger & Bohlen, (1999) Cancer & Metastasis Rev. 18: 411-419). Their use has been demonstrated in both the diagnostic and therapeutic fields by their ability to recruit new effector functions or to target some molecules on the surface of tumor cells. Where bispecific antibodies are to be used, these may be conventional bispecific antibodies that can be produced in various ways (Holliger & Winter, (1993) Curr. Op. Biotech. 4: 446-449). An example of a bispecific antibody is that of BiTE® technology (Micromet, Inc.) which can be linked directly via a short flexible peptide using the binding domains of two antibodies with different specificities. It can be mentioned. It combines two antibodies on a short single polypeptide chain. Diabodies and scFv can be constructed without the Fc region, using the variable domain alone, potentially reducing the effects of the anti-idiotype reaction.

Bispecific antibodies can be constructed as whole IgG, as bispecific F (ab ') _2, as Fab'PEG, as diabodies, and as bispecific scFv. Furthermore, two bispecific antibodies can be linked using routine methods known in the art to form tetravalent antibodies. Bispecific diabodies, as opposed to bispecific whole antibodies, may be particularly useful because they can be readily constructed and expressed in E. coli.

  As mentioned above, in some embodiments, binding members according to the present invention are antibodies that bind c-MAF. High potency binding members can be obtained directly from the initial screening. Assays and potencies are described in more detail elsewhere herein.

  In some embodiments, the binding member is an antigen binding molecule or fragment thereof that binds human c-MAF, wherein the antibody binding molecule or fragment thereof comprises at least about 70% of the amino acid sequence of SEQ ID NO: 38, About 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical heavy chain CDR1 and / or at least about 70% with the amino acid sequence of SEQ ID NO: 40, about 75 %, About 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical heavy chain CDR2 and / or at least about 70%, about 75% with the amino acid sequence of SEQ ID NO: 42 About 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical heavy chain CDR3; and / or the amino acid sequence of SEQ ID NO: 26 Light chain CDR1 at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical to the light chain CDR1 and / or at least with the amino acid sequence of SEQ ID NO: 28 About 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical light chain CDR2 and / or at least about 70 with the amino acid sequence of SEQ ID NO: 30 %, About 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% identical light chain CDR3s.

  In some embodiments, the antigen binding molecule or fragment thereof is an antibody. In some embodiments, the antibody is a rabbit antibody, a mouse antibody, a chimeric antibody or a humanized antibody.

In some embodiments, the antibody or fragment thereof is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 70% It comprises V _H domains having a sequence of about 99%, at least about 99%, or at least about 100% identity.

In some embodiments, the antigen binding molecule or fragment thereof has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of the amino acid sequence of SEQ ID NO: 21. %, comprising a _{V L} domain having at least about 99%, at least about 99%, or at least about 100% identical sequences.

  In some embodiments, the antibody or fragment thereof has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, with the amino acid sequence of SEQ ID NO: 16. It comprises heavy chain sequences at least about 99%, at least about 99%, or at least about 100% identical.

  In some embodiments, the antibody or fragment thereof has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, amino acid sequence with SEQ ID NO: 20 At least about 99%, at least about 99%, or at least about 100% identical light chain sequences.

In some embodiments, an antibody _VH variable domain having the amino acid sequence of the selected binding member can be provided in an isolated form, such as a binding member comprising such a _VH domain. In some embodiments, an antibody _VL variable domain having the amino acid sequence of the selected binding member can be provided in an isolated form, as a binding member comprising such a _VL domain. In some embodiments, the binding member is a variant of any binding member disclosed herein. In some embodiments, the V _H domain and / or V _L domain is a variant of any V _H domain and / or V _L domain disclosed herein.

  The ability to bind c-MAF can be further tested, as well as the ability to compete with any of the antibody molecules of the invention for binding to c-MAF. The binding affinities of different binding members can be compared under appropriate conditions.

Variants of the V _H and V _L domains and CDRs of the invention, including those whose amino acid sequences are set forth herein and which can be used for the binding members of the invention, alter or mutate the sequence, It can be obtained by a method of screening for antigen binding members having desired characteristics. Examples of desired features are:
· Increased binding affinity to antigen compared to known antibodies specific for the antigen · Neutralization of said activity compared to known antibodies specific for the antigen, if the antigen activity is known The ability to immunoprecipitate a specific competitive ability with a known antibody or ligand to an antigen at an increased specific ratio, specific epitope such as a linear epitope (eg linear and / or restricted) These include the ability to use conformationally screened peptides) or conformational epitopes formed by non-contiguous residues, including the ability to modulate new biological activities or downstream molecules of c-MAF. It is not limited to. Such methods are also provided herein.

  In some embodiments, the binding member binds to the antigen c-MAF. In some embodiments, the binding member binds human c-MAF. In some embodiments, the binding member binds to an epitope corresponding to 83-EQKAHLEDYYWMTGYPQQ-100 (18a.a.) (SEQ ID NO: 22) of c-MAF of human origin.

The antigen binding site of an antibody comprised of a V _H domain and a V _L domain is typically as long as the six loops of the polypeptide (three from the light chain variable domain (V _L ) and the heavy chain variable domain (V _H) 3) derived from Analysis of antibodies of known atomic structure has elucidated the relationship between the sequence of the antibody combining sites and the three-dimensional structure. These relationships mean that, except for the third region (loop) in the V _H domain, the binding site loop has one of a small number of main-chain conformations or standard structures. The canonical structure formed in a particular loop has been shown to be determined by its size and the presence of particular residues at key sites in both the loop and framework regions (Chothia et al., (1992) J. Molecular Biology 227: 799-817; Al-Lazikani et al., (1997) J. Mol. Biol. 273 (4): 927-948).

  This study of sequence-structure relationships is the residue of an antibody whose sequence is known but whose three-dimensional structure is unknown, and which is important for maintaining the three-dimensional structure of its CDR loops, thus maintaining binding specificity. It can be used to predict residues. In the structural approach, any free available or commercially available package such as WAM (Whitelegg & Rees, (2000) Prot. Eng. 12: 815-824) can be used to create models of antibody molecules (Chothia) (1986) Science 223: 755-758). Then, a protein visualization and analysis software package such as Insight II (Accelrys, Inc.) or Deep View (Guex & Peitsch, (1997) Electrophoresis 18: 2714-2723) is used to evaluate possible substitution of each position in CDR It can. This information can then be used to make substitutions that may have minimal or beneficial effects on activity.

The techniques required to make substitutions within amino acid sequences of CDRs, antibody _VH domains or _VL domains and binding members are generally available in the art. Create variant sequences with substitutions (which may or may not be presumed to have minimal or beneficial effects on activity), for c-MAF binding ability, and / or It may be tested for any other desired property.

Variable domain amino acid sequence variants of any of the V _H and V _L domains whose sequences are specifically disclosed herein may be used in accordance with the present invention as discussed.

A further aspect of the invention relates to the _VH domain of at least about 60, about 70, about 80, about 85, about 85, about 90, about 95 of the antibodies shown in the attached sequence listing (e.g. SEQ ID NO: 17). comprises about 98 or V _H domain having about 99% amino acid sequence identity, and any / or accompanying the antibody in the sequence listing (e.g., SEQ ID NO: 21) V _L domains with at least about 60, about 70, about 80, about 85, about 90, about 95, is an antibody molecule comprising a _{V L} domain having about 98 or about 99% amino acid sequence identity. As an algorithm that can be used to calculate the percent identity of two amino acid sequences, for example, using default parameters, such as BLAST (Altschul et al. (1990) J. Mol. Biol. 215 (3): 403-10 And FASTA (Pearson & Lipman, (1988) PNAS USA 85 (8): 2444-8), or Smith-Waterman algorithm (Smith & Waterman, (1981) J. Mol. Biol. 147 (1): 195-7). . Certain variants may include one or more amino acid sequence changes (additions, deletions, substitutions, and / or insertions of amino acid residues).

  Changes can be made in one or more framework regions and / or one or more CDRs. As changes do not usually result in loss of function, binding members comprising such altered amino acid sequences may retain the ability to bind c-MAF. It may retain the same quantitative binding ability as a non-altered binding member, for example as measured in the assays described herein. Binding members comprising such altered amino acid sequences may have improved c-MAF binding ability.

  The change may be replacing one or more amino acid residues with non-naturally occurring or non-standard amino acids, modifying one or more amino acid residues into non-naturally occurring or non-standard forms, or Alternatively, it may involve inserting more non-naturally occurring or non-standard amino acids into the sequence. Examples of the number and location of changes in the sequences of the invention are described elsewhere herein. As naturally occurring amino acids, G, A, V, L, I, M, P, F, W, S, T, N, Q, Y, C, K, R, H, and the like according to their standard single-letter codes There are 20 "standard" L-amino acids identified as D, E. Non-standard amino acids include any other residue that may be incorporated into the polypeptide backbone or may result from modification of existing amino acid residues. Non-standard amino acids may be naturally occurring or non-naturally occurring. Several naturally occurring non-standard amino acids are known in the art such as 4-hydroxyproline, 5-hydroxylysine, 3-methylhistidine, N-acetylserine (Voet & Voet, Biochemistry, 3rd Edition, (Wiley) 2004). Only those amino acid residues that are derivatized at their N-alpha position are located at the N-terminus of the amino acid sequence. Usually, in the present invention, the amino acid is an L-amino acid, but it may be a D-amino acid. Thus, the change may comprise altering the L-amino acid to a D-amino acid or replacing it with a D-amino acid. Methylation, acetylation and / or phosphorylation forms of amino acids are also known, and the amino acids in the present invention can be subjected to such modifications.

  The amino acid sequences in the antibody domains and binding members of the invention may comprise the non-naturally occurring or non-standard amino acids described above. Non-standard amino acids (eg, D-amino acids) may be incorporated into the amino acid sequence during synthesis, or by modification or replacement of the "original" standard amino acids during synthesis of the amino acid sequence.

  The use of non-standard and / or non-naturally occurring amino acids increases structural and functional diversity, and thus can increase the likelihood of achieving the desired c-MAF binding properties in binding members of the invention. Furthermore, D-amino acids and analogs have different pharmacokinetic profiles compared to standard L-amino acids after administration to animals (eg, humans) due to in vivo degradation of polypeptides with L-amino acids It has been shown that this means that D-amino acids are advantageous for some in vivo applications.

The novel V _H or V _L regions carrying CDR derived sequences of the invention are within the entire variable domain using random mutagenesis of one or more of the selected V _H and / or V _L genes. Can be generated by generating mutations. Such techniques are described by Gram et al. (1992) using error prone PCR. In some embodiments, one or two amino acid substitutions are made within a set of all variable domains or CDRs. Another method that may be used is to direct mutagenesis on the CDR regions of the V _H or V _L genes (Barbas et al. (1994) PNAS USA 91: 3809-3813; Schier et al. (1996) J. Mol. Mol. Biol. 263: 551-567).

  All the above techniques are themselves known in the art, and one of ordinary skill in the art would use such techniques to provide binding members of the invention using routine methods in the art. Can.

A further aspect of the invention is a method for obtaining the antigen binding site of an antibody against c-MAF, which comprises the addition, deletion of one or more amino acids in the amino acid sequence of the V _H domain as indicated herein. Providing the _VH domain, which is an amino acid sequence variant of the _VH domain, by deletion, substitution or insertion, optionally combining the thus provided _VH domain with one or more _VL domains, And one or more combinations of V _H domains or V _H / V _L are binding members to c-MAF or antigen binding sites of the antibody, wherein one or more desired properties (eg, , C-MAF binding ability, and a method comprising the step of identifying the antigen binding site of the binding member or antibody . The V _L domain may have the amino acid sequence substantially as set forth herein. Similar methods can be used to combine one or more sequence variants of the V _L domain disclosed herein with one or more V _H domains. In some embodiments, variants of the V _L domain or V _H domain are functional variants. As mentioned above, the CDR amino acid sequences substantially as set forth herein may be retained as CDRs in a human antibody variable domain or a substantial portion thereof. The HCDR3 sequences substantially as set forth herein represent embodiments of the present invention, each of which may be retained as HCDR3 or a substantial portion thereof in a human heavy chain variable domain.

Similarly, one or more or all three CDRs can be grafted into the repertoire of V _H or V _L domains and then screened for binding member (s) of c-MAF .

For example, one or more of the antibodies HCDR1, HCDR2 and HCDR3 or a set of HCDRs (SEQ ID NO: 38, 40 and 42) may be used and / or one or more of the antibodies LCDR1, LCDR2 and LCDR3, or A set of LCDRs (SEQ ID NOs: 26, 28 and 30) may be used. Similarly, other _VH and _VL domains, sets of CDRs, and sets of HCDRs and / or sets of LCDRs disclosed herein may be used.

  Other engineering steps introduce linkers to include antibody constant regions, other variable domains, or detectable / functional labels, as discussed in more detail elsewhere herein. Including connecting the variable domain of the invention to additional protein sequences.

In some embodiments of the invention, the binding member comprises a set of V _H and V _L domains, but a single binding domain based on either V _H or V _L domain sequences is an additional feature of the invention Form an aspect. It is known that single immunoglobulin domains, in particular _VH domains, can bind to target antigens in a specific way. In the case of any of the single binding domains, these domains can be used to screen for complementary domains capable of forming a two domain binding member capable of binding c-MAF. This is a phage display using the so-called hierarchical dual combinatorial approach disclosed in International Patent Application Publication WO 92/01047 (McCafferty et al.) And Marks et al. (1992) Biotechnology 10 (7): 779-83. It can be achieved by screening methods.

Binding members of the invention may further comprise antibody constant regions or parts thereof, such as human antibody constant regions or parts thereof. For example, the V _L domain may be attached at its C-terminus to an antibody light chain constant domain. Similarly, binding members based on the V _H domain are immunized at their C-terminal end from any antibody isotype (eg IgG, IgA, IgE, and IgM and any of the isotype subclasses, in particular IgG ₁ and IgG ₄ ) It may be linked to all or part of a globulin heavy chain (eg, a C _H 1 domain). IgG ₁ is advantageous due to its effector function and ease of manufacture. Any synthetic or other constant region variant that has these properties and stabilizes the variable region may also be useful in the present invention.

  Binding members of the invention may also include antibodies or fragments comprising a modified Fc region that comprises at least one amino acid modification relative to a wild type Fc region. Variant Fc regions can be designed to bind to Fc receptors with greater or less affinity compared to comparable molecules comprising wild type Fc regions. Fc region refers to a naturally occurring or synthetic polypeptide homologous to the IgG C-terminal domain produced by papain digestion of IgG. IgG Fc has a molecular weight of approximately 50 kD. In the antibodies and / or fragments of the invention, only the entire Fc region or half-life enhancing moiety may be used.

The Fc region can optionally be mutated to fix complement and to inhibit its ability to bind with high affinity to Fc receptors. In one embodiment of the invention, the antibody or fragment increases the half life of the antibody or fragment in the biological environment, such as serum half life, or half life as measured by in vitro assays, of the naturally occurring Fc region. Can be provided with a modified Fc region that has been modified to Methods for altering the original morphology of IgG Fc regions are also described in US Pat. No. 6,998,253 (Presta & Snedecor). Examples of effector functions that can be altered (eg, enhanced) by altering the Fc region by altering the glycosylation pattern or by altering the amino acid sequence of the Fc region include, but are not limited to, eg, complement These include dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Possible modifications include: substitution with alanine, conservative substitution, non-conservative substitution, or substitution with the corresponding amino acid residue at the same position from different IgG subclasses (eg, by the corresponding IgG ₂ residue at that position) Insertions, deletions or substitutions of one or more amino acid residues (including replacement of IgG ₁ residues).

  Thus, in a further aspect, the invention is a c-MAF binding member as described elsewhere herein, wherein at least IgG is modified to increase one or more effector functions. The Fc region containing the CH2 region or c-MAF binding member containing the equivalent region is included. In one embodiment, the binding member is modified to alter the glycosylation pattern of the N-linked oligosaccharides to increase the activity of one or more effector functions. In another embodiment, the binding member is altered such that the amino acid sequence of the Fc region is altered to increase the activity of one or more effector functions. Methods of measuring effector function activity and determining whether they increase are well known in the art.

  Binding members of the invention may be labeled with a detectable or functional label. Thus, binding members or antibody molecules may be present in the form of immunoconjugates in order to obtain a detectable and / or quantifiable signal. An immunoconjugate may comprise an antibody molecule of the invention conjugated to a detectable or functional label. The label may be any molecule capable of generating or being induced to generate a signal, including but not limited to fluorochromes, radiolabels, enzymes, chemiluminescers, or photosensitizers. . Thus, binding can be detected and / or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity, or light absorbance. Illustrative examples of labels that may be used include radioactive isotopes, enzymes, fluorochromes, chemiluminescent reagents, enzyme substrates or cofactors, enzyme inhibitors, particles, dyes and the like.

  Suitable labels include, but are not limited to, enzymes such as, but not limited to, alkaline phosphatase, glucose-6-phosphate dehydrogenase ("G6PDH"), alpha-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, Lysozyme, malate dehydrogenase, and peroxidase, such as horseradish peroxidase; dyes; fluorescent labels or fluorescent dyes, such as fluorescein and its derivatives, rhodamine compounds and derivatives, green / yellow fluorescent protein (G / YFP), red fluorescent protein (RFP) , Blue fluorescent protein (BFP), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and others Fluorescens; fluorophores such as lanthanide cryptates and chelates such as europium (Perkin Elmer and Cis Biointernational), chemiluminescent labels or chemiluminescers such as isoluminol, luminol and dioxetanes; bioluminescent labels such as luciferase and luciferin Sensitizer: coenzyme; enzyme substrate: bromine 77, carbon 14, cobalt 57, fluorine 8, gallium 67, gallium 68, hydrogen 3 (tritium), indium 111, indium 113 m, iodine 123 m, iodine 125, iodine 126, Iodine 131, iodine 133, mercury 107, mercury 203, phosphorus 32, rhenium 99 m, rhenium 101, rhenium 105, ruthenium 95, ruthenium 97, ruteni 103, ruthenium 105, scandium 47, selenium 75, sulfur 35, technetium 99, technetium 99m, tellurium 121m, tellurium 122m, tellurium 125m, thulium 165, thulium 167, thulium 168, yttrium 199, and the like mentioned herein. Radiolabels, including but not limited to: particles, such as latex particles or carbon particles; metal sols; crystallites; liposomes; may be further labeled with dyes, catalysts, or other detectable groups Molecules, such as biotin, digoxigenin, or 5-bromodexirizine; toxin moieties, such as Pseudomonas exotoxin (PE or its cytotoxic fragment or variant thereof) diphtheria toxin or its cytotoxicity Fragments or botulinum toxins A, B, C, D, E, or F, ricin or cytotoxic fragments thereof, such as ricin A, abrin or cytotoxic fragments thereof, saporin or cytotoxic fragments thereof, pokeweed antiviral Sex toxins or cytotoxic fragments thereof, and toxin moieties selected from the group of bryodin 1 or cytotoxic fragments thereof, and the like.

  Suitable enzymes and coenzymes are disclosed in U.S. Patent No. 4,275,149 (Litman et al.) And U.S. Patent No. 4,318,980 (Boguslaski et al.), Suitable fluorescent agents and chemiluminescers are No. 4,275,149, which are incorporated herein by reference in their entirety. The label may further be detected via binding to a specific detectable homologous moiety, such as labeled avidin or streptavidin, or genetically engineered streptavidin, such as streptactin (IBA GmbH, Gottingen, DE) Chemical moieties such as biotin. Detectable labels can be attached to the antibodies of the invention using conventional chemistry known in the art.

  Immunoconjugates or their functional fragments can be prepared by methods known to those skilled in the art. They are either directly or via spacer or linking groups such as polyaldehydes such as glutaraldehyde, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DPTA) or as described above for coupling agents such as therapeutic conjugates. The enzyme or fluorescent label may be attached in the presence of what is mentioned in. Conjugates containing a fluorescein-type label can be prepared by reaction with isothiocyanate.

  The existing methods known to the person skilled in the art for coupling therapeutic radioisotopes directly or to an antibody via a chelating agent such as EDTA, DTPA as described above are used for radioactive elements which can be used for diagnosis It can be done. Similarly, with iodine-131 by the chloramine T method (Hunter & Greenwood, (1962) Nature 194: 495-6), or by the technique otherwise described in US Patent 4,424, 200 (Crockford & Rhodes), or Technetium-99m (Tc-99m) linked via DTPA as described in US Pat. No. 4,479,930 (Hnatowich), both of which are herein incorporated by reference in their entirety. It is possible to carry out labeling.

  There are numerous ways in which the label can produce a signal detectable by external means, for example by visual inspection, electromagnetic radiation, heat and chemical reagents. The label may also be attached to another binding member that binds to the binding member of the invention or to a support.

  As the label may generate the signal directly, no additional components are required to generate the signal. Many organic molecules, such as fluorescent agents, can absorb ultraviolet and visible light, and light absorption transfers energy to these molecules, raising them to an excited energy state. This absorbed energy is then dissipated by the emission of light of the second wavelength. The radiation at this second wavelength can also transfer energy to the labeled acceptor molecule, and the resulting energy can be dissipated from the acceptor molecule by light emission, such as fluorescence resonance energy transfer (FRET). Other labels that directly generate a signal include radioactive isotopes and dyes.

  Alternatively, the label may require another component to generate a signal, so that the signal generation system comprises a substrate, a coenzyme, an enhancer, an additional enzyme, a substance that reacts with the enzyme product, a catalyst, an activator, a cofactor, Includes all components necessary to generate a measurable signal, which may include inhibitors, scavengers, metal ions, and specific binding agents necessary for the binding of the signal producing substance. A detailed discussion of suitable signal generation systems can be found in US Pat. No. 5,185,243 (Ullman et al.). The present invention provides a method comprising the step of causing or allowing the binding of a binding member provided herein specific for c-MAF. As mentioned, such binding may occur, for example, in vivo after administration of the binding member or a nucleic acid encoding the binding member, or it may be, for example, ELISA, Western blotting, affinity chromatography, immunocytochemistry, immunization Precipitation, neutralization, and in vitro may occur in biochemical or cell based assays.

Determining Levels of Genes of Interest Using Antibodies of the Invention In a preferred embodiment, binding members (eg, antibodies) of the invention are used to quantify c-MAF protein expression levels. c-MAF protein expression levels can be quantified by any conventional method that allows for the detection and quantification of said proteins in a sample from a subject. As a non-limiting illustration, the above protein levels can be determined, for example, by using an antibody (or a fragment thereof containing an antigenic determinant) capable of binding to c-MAF, followed by quantifying the complex formed. It can be quantified. In some embodiments, the antibody used to detect c-MAF protein expression levels is any antibody described herein. The antibodies used in these assays may or may not be labeled. There are a wide variety of known assays that can be used in the present invention using unlabeled antibody (primary antibody) and labeled antibody (secondary antibody); these techniques include Western blot or Western transfer, ELISA (enzyme Includes coupled immunosorbent assay), RIA (radioimmunoassay), competitive EIA (competitive enzyme immunoassay), DAS-ELISA (double antibody sandwich ELISA), immunocytochemical and immunohistochemical techniques, specific antibodies Techniques based on the use of protein microarrays or biochips, or assays based on colloidal precipitation in the form of dipsticks and the like. Other methods for detecting and quantifying the c-MAF protein include affinity chromatography, ligand binding assays and the like. When immunological methods are used, any antibody or reagent known to bind with high affinity to c-MAF protein can be used to detect that amount. Nevertheless, antibodies, such as polyclonal sera, hybridoma supernatants or monoclonal antibodies, antibody fragments, Fv, Fab, Fab 'and F (ab') 2, scFv, humanized diabodies, triabodies, The use of tetrabodies, nanobodies, alphabodies, stapled peptides, cyclopeptides and antibodies is preferred. In some embodiments, the antibody is INB-1-11-8 as described in Example 1. The INB-1-11-8 light chain sequence is SEQ ID NO: 20 and the INB-1-11-8 heavy chain sequence is SEQ ID NO: 16.

K _D can be determined by surface plasmon resonance, such as BIAcore®. Surface plasmon resonance, for a ligand is bound to a solid support, passed through a analyte in a fluid phase, the association rate between the analyte and the ligand (k _a) and dissociation rates (k _d) determined To do. Surface plasmon resonance can be performed, for example, by passing the binding member in the fluid phase against gB protein bound to a support. Biacore allows one to determine the extent to which different molecules interact with a single partner immobilized on the sensor surface, revealing the specificity of the interaction. Biacore allows the determination of the association rate between the analyte and the ligand _{(k a)} and dissociation rates _{(k d).} Kinetics of the interaction, i.e. the rate of formation of the complex (k _a) and dissociation (k _d) can be determined from the information of the sensorgrams. The affinity is expressed as the dissociation constant K _D (which is calculated from the ratio of dissociation rate and association rate constant k _d / k _a determined by surface plasmon resonance using the monovalent analyte data model) obtain. In some embodiments, the affinity is a monovalent binding affinity.

  In some embodiments, the antibodies or fragments thereof described herein are at least about 1 μM, 100 nM, 50 nM, 10 nM, at least about 9 nM, at least about 8 mM, at least about 7 nM, at least about 6 nM, at least about 5 nM At least about 4 nM, at least about 3 nM, at least about 2.5 nM, at least about 2 nM, at least about 1.9 nM, at least about 1.8 nM, at least about 1.7 nM, at least about 1.6 nM, at least about 1.5 nM, at least about About 1.4 nM, at least about 1.3 nM, at least about 1.2 nM, at least about 1.1 nM, at least about 1.0 nM, at least about 0.9 nM, at least about 0.8 nM, at least about 0.7 nM, at least about 0 .6 nM, at least about 0 5 nM, at least about 0.4 nM, at least about 0.3 nM, at least about 0.2 nM, at least about 0.1 nM, at least about 75 pM, at least about 50 pM, at least about 25 pM, or at least about 1 pM affinity (KD), It binds to human c-MAF. In some embodiments, the affinity (KD) of the antibody for c-MAF is at least about 1 nM to about 1.2 nM, at least about 1 nM to about 1.5 nM, at least about 1 nM to about 2.0 nM, at least about 1 nM To about 3.0 nM, at least about 1 nM to about 4.0 nM, at least about 1 nM to about 5.0 nM, at least about 1 nM to about 6.0 nM, at least about 1 nM to about 7.0 nM, at least about 1 nM to about 8.0 nM , At least about 1 nM to about 9.0 nM, at least about 1 nM to about 10 nM, and in some embodiments, at least about 1 nM to about 50 nM, at least about 1 nM to about 100 nM, at least about 1 nM to about 1 μM, at least about 0.1 nM To about 1.5 nM, or at least about 10 pM to about 1.5 nM. In some embodiments, the antibodies described herein or fragments thereof bind human c-MAF with an affinity (KD) of at least about 1.1 nM.

  In certain embodiments, c-MAF protein levels are quantified by Western blot, ELISA or protein array.

In another specific embodiment, c-MAF protein levels are quantified from exosomes, circulating DNA or circulating tumor cells. Exosomes are 40-100 nm membrane vesicles secreted by most cell types in vivo and in vitro. Exosomes form as a specific population of endosomes called multivesicular bodies (MVB) by budding inward into the lumen of the compartment. When the MVB and plasma membrane fuse, these internal vesicles are secreted. Exosomes can be isolated from a wide variety of cell lines or fluids by several methods well known in the art (Thery C. et al., Curr Protoc Cell Biol. 2006 Apr; Chapter 3: Unit 3.22) (all of which The contents of which are incorporated herein by reference). ExoQuick ^TM or some commercial kits such ExoTest ^TM are available for isolating exosomes.

  The invention provides a method for directly measuring the level of an antigen, for example by using a binding member according to the invention in a biosensor system. For example, the invention is a method of detecting and / or measuring binding to c-MAF, comprising the steps of (i) exposing said binding member to c-MAF, and (ii) said binding to c-MAF Detecting binding of members, including any of the methods described herein or methods of detecting binding using a detectable label. This and any other binding detection methods described herein may be interpreted directly by the practitioner of the method, for example by visually observing the detectable label. Alternatively, this method or any other binding detection method described herein may be an autoradiographic, photographic, computer printed out, flow cytometric report, diagram, chart, test tube or container containing results or A report may be generated in the form of a well or any other visual or physical representation of the results of the method.

  The amount of binding of the binding member to c-MAF can be determined. The quantification can be related to the amount of antigen in the test sample that can be diagnosed. Screening for binding and / or quantitation thereof, for example, screens patients for the disease or disorder referred to herein and / or any other disease or disorder associated with aberrant c-MAF expression and / or activity. It can be useful.

  The diagnostic method of the invention comprises the steps of (i) obtaining a tissue or fluid sample from the subject, (ii) exposing the tissue or fluid sample to one or more binding members (eg, antibodies) of the invention And (iii) detecting the bound c-MAF as compared to the control sample, wherein the increase in the amount of c-MAF binding compared to the control is c-MAF expression and / or activity May be included. Tissue or fluid samples to be tested include tumors, blood, serum, saliva, urine, sputum, biopsy material, or any tissue suspected of containing Maf. Subjects who are positive for an increase in c-MAF in the test may also benefit from the treatment methods disclosed later herein. One of ordinary skill in the art, in view of the methods disclosed herein, can select an appropriate manner of determining binding of a binding member to an antigen according to their preferences and general knowledge.

  One embodiment comprises, in the second step, comparing the c-MAF gene expression level obtained in a sample from the subject (eg, a tumor sample) with a reference value.

  Once the c-MAF gene expression level in a sample from a subject with cancer is measured and compared to a reference value, the expression level of said gene is elevated compared to said reference value If so, it can be concluded that the subject is more prone to develop metastases (eg, bone metastases).

  Determination of c-MAF gene expression levels should correlate with the reference value.

  In one embodiment, the reference value (s) as intended herein may represent an absolute amount of MAF. In another embodiment, the amount of any one or more biomarkers in a sample from the subject being tested may be determined by direct comparison to a reference value (eg, increase or decrease or increase factor or Regarding the reduction factor). Advantageously, this allows the amount of any one or more biomarkers in a sample from a subject to be determined without first determining the absolute amount of each of the one or more biomarkers. It may be possible to compare to a reference value (in other words to measure the relative amount of any one or more biomarkers in the sample from the subject relative to the reference value).

  In a preferred embodiment, the reference value is c-MAF gene expression level in a control sample or reference sample. Depending on the type of tumor to be analyzed, the exact nature of the control or reference sample may vary. Thus, where prognosis is to be assessed, the reference sample is measured in a tumor tissue collection of non-metastasized cancer samples or biopsy samples from subjects with non-metastasized cancers. It is a sample corresponding to the median of c-MAF gene expression level.

  The reference sample is typically obtained by combining equal volumes of samples from a population of subjects. In general, representative reference samples are obtained from subjects that are clinically well documented and those that are well characterized as having no metastases. In such samples, the normal concentration (reference concentration) of the biomarker (MAF gene) can be determined, for example, by providing the mean concentration of the reference population. Various considerations are made in determining the reference concentration of the marker. Such considerations include the patient's age, weight, gender, general physical condition and the like. For example, preferably according to the above considerations, eg, classified according to various age categories, equivalent amounts of at least about 2, at least about 10, at least about 100 to preferably more than about 1000 A group of subjects is considered as a reference group. The sample set from which the reference level is derived is preferably formed by a subject suffering from the same type of cancer as the subject subject in the study.

  In certain embodiments, the reference value for "elevated" or "reduced" expression of c-MAF expression is from a subject whose disease has been sufficiently documented by any of the methods mentioned above. It is determined by calculating the percentile of c-MAF expression levels by conventional means including performing the assay on one or several samples isolated. The "reduced" level of c-MAF is then preferably less than about 50 percentile of c-MAF expression levels in the normal population (eg, less than about 60 percentile in the normal population, less than about 70 percentile in the normal population, It may be assigned to a sample (including an expression level which is about 80 th or lower in the normal population, about 90 th lower or lower in the normal population and about 95 th lower or lower in the normal population. The "elevated" c-MAF gene expression level is then preferably such that the c-MAF gene expression level is at least the 50 th percentile in the normal population (eg, about 60 th percentile or more in the normal population, about 70 th percentile or more in the normal population Can be assigned to samples containing expression levels that are greater than about 80 percentile in the normal population, greater than about 90 percentile in the normal population and greater than about 95 percentile in the normal population.

The reactivities of binding members in a sample can be determined by any suitable means. Competition binding assays use radioactive antigens, such as isotope labels such as ⁹⁹ Tc, ¹⁴ C, ¹³¹ I, ¹²⁵ I, ³ H, ³² P or ³⁵ S, or antigens or analogues linked to a reporter molecule It can be used with non-radioactive antigens. The reporter molecule can be a fluorescent dye, a phosphor, or a laser dye having spectrally separated absorption or emission characteristics. Suitable fluorescent dyes include fluorescein, rhodamine, phycoerythrin, and Texas Red as well as lanthanide chelates or cryptates. Suitable chromogenic dyes include diaminobenzidine.

  Other reporters include polymeric colloidal particles or particulate matter, such as colored, magnetic or paramagnetic latex beads, and direct or indirect detection of detectable signals for visual observation, electronic detection or Included are biologically or chemically active agents that allow for alternative recording. These molecules can be, for example, enzymes that catalyze reactions that generate or change color or cause changes in electrical properties. They can be molecularly excitable, and electronic transitions between energy states result in characteristic spectral absorption or emission. They may include chemical entities used in conjunction with biosensors. Biotin / avidin or biotin / streptavidin and alkaline phosphatase or horseradish peroxidase detection systems can be used.

  The signals generated by the individual binding member-reporter conjugates can be used to obtain quantifiable absolute or relative data of the binding of the relevant binding member in the sample (normal and test).

  The invention also relates to the use of the above binding member (eg antibody) for measuring antigen levels (eg Maf) in a competition assay (in other words by using the binding member provided by the invention in a competition assay) Provide a method of measuring the level of antigen in a sample). This may be the case when physical separation of bound antigen from unbound antigen is not required. Linkage of the reporter molecule to the binding member is one possibility, as physical or optical changes occur upon binding. The reporter molecule may generate a detectable signal, which may be quantifiable, directly or indirectly. The linkage of reporter molecules may be direct or indirect, covalent (eg via a peptide bond) or non-covalent. Linkage via a peptide bond may be the result of recombinant expression of a gene fusion encoding an antibody and a reporter molecule.

  In various aspects and embodiments, the invention extends to binding members that compete with any antibody for binding to c-MAF. Competition between binding members may, for example, tag a particular reporter molecule to one binding member (which may be detected in the presence of the other non-tagged binding member (s)) to bind the same epitope or It can be assayed in vitro by allowing the identification of binding members that bind to overlapping epitopes. The competition uses, for example, an ELISA in which hCMV is immobilized on a solid phase and a first tagged or labeled binding member is added to the solid phase along with one or more other non-tagged or unlabeled binding members. Or by surface plasmon resonance. The presence of untagged binding member in competition with tagged binding member is observed by the reduction of the signal emitted by the tagged binding member.

  For example, the present invention is a method of identifying a c-MAF binding compound comprising the steps of: (i) immobilizing a protein on a support, (ii) said immobilized protein and at least one tag according to the invention or Contacting the labeled binding member and one or more untagged or unlabeled test binding compounds simultaneously or stepwise, and (iii) observing a reduction in the amount of binding tag from the tagged binding member Thereby comprising a method comprising the step of identifying new c-MAF binding compounds. Such methods may be implemented in a high throughput manner, using a multiwell format or an array format. Such assays can also be performed in solution. See, eg, US Pat. No. 5,814,468 (Sliman et al.), Which is incorporated herein by reference in its entirety. As mentioned above, the detection of binding can be interpreted directly by the practitioner of the method, for example by visually observing a decrease in the detectable label or its presence. Alternatively, the binding method of the invention may generate a report in the form of an autoradiographic, photographic, computer printout, flow cytometric report, or any other visual or physical representation of the result of the method.

  Competition assays can also be used for epitope characterization. In one example, epitope characterization can be used to identify the epitope to which a c-MAF binding member (which may optionally have optimized neutralizing and / or regulatory properties) has bound. Such epitopes may be linear or conformational epitopes. The conformational epitope may comprise at least two different domains of Maf, wherein the c-MAF protein is folded into its three-dimensional or four-dimensional structure to provide an inhibitor of Maf (eg, provided herein When forming conformational epitopes recognized by any c-MAF binding member, the domains are located in close proximity to one another. For testing for competition, peptide fragments of the antigen may be used, in particular peptides which comprise or consist of the epitope of interest. Peptides having an epitope sequence and one or more amino acids at either end may be used. Binding members according to the invention may be such that their binding to an antigen is inhibited by a peptide having or comprising a predetermined sequence.

Methods of Using Binding Members Described Herein In some embodiments, the present invention provides a test using a binding member (eg, an antibody) described herein, variants or fragments thereof. In vitro methods for quantifying c-MAF gene expression levels in body tumor samples are directed.

In some embodiments, the present invention is an in vitro method for the diagnosis of metastasis in a subject having cancer and / or for the prognosis of a tendency to metastasize in a subject having cancer. ,
(I) quantifying c-MAF gene expression levels in a tumor sample of said subject using a binding member (e.g. an antibody), variant or fragment thereof as described herein and (ii) Comparing the expression level obtained in (i) with the expression level of c-MAF gene in a control sample,
Here, if the expression level of c-MAF gene in the tumor sample is elevated compared to the expression level of c-MAF gene in the control sample, then the subject has a positive diagnosis for metastasis. It is directed to in vitro methods that have or are more prone to metastasis.

In some embodiments, the present invention is an in vitro method for designing a personalized treatment for a subject having cancer,
(I) quantifying c-MAF gene expression levels in a tumor sample of said subject using a binding member (e.g. an antibody), variant or fragment thereof as described herein and (ii) Comparing the expression level obtained in (i) with the expression level of c-MAF gene in a control sample,
Wherein said subject is capable of receiving treatment aimed at preventing and / or treating metastasis when said expression level is elevated compared to said reference value I assume. If the expression level is not elevated compared to the reference value, the subject is not able to receive treatment aiming for the prevention and / or treatment of metastasis. In some embodiments, the sample is a tumor sample, a circulating tumor sample, a circulating tumor DNA, or a tumor derived sample comprising tumor derived exosomes.

In some embodiments, the present invention is an in vitro method for designing a personalized therapy for a subject with cancer with metastasis, comprising
(I) quantifying c-MAF gene expression levels in a tumor tissue sample of said subject using a binding member (eg, an antibody), variant or fragment thereof as described herein; ii) comparing the expression level obtained in step (i) with the expression level of c-MAF gene in a control sample,
Here, when c-MAF gene expression level in the tumor tissue sample is elevated compared to the expression level of c-MAF gene in the control sample, the subject prevents or inhibits bone metastasis and degradation. It is directed to in vitro methods capable of receiving the desired treatment. If the expression level is not elevated compared to the reference value, the subject is not able to receive treatment aiming for prevention and / or treatment of bone metastasis and degradation.

In some embodiments, the present invention is an in vitro method for designing a personalized therapy for a subject with cancer with metastasis, comprising
(I) quantifying c-MAF gene expression levels in a bone metastatic tumor tissue sample of said subject using a binding member (eg, antibody), variant or fragment thereof as described herein And (ii) comparing the expression level obtained in step (i) with the expression level of c-MAF gene in a control sample,
Here, when the c-MAF gene expression level in the tumor tissue sample is elevated compared to the c-MAF gene expression level in the control sample, the subject aims to prevent or inhibit bone degradation. The present invention is directed to in vitro methods that are capable of receiving treatment. If the expression level is not elevated compared to the reference value, the subject is not able to receive treatment aiming for the prevention and / or treatment of bone degradation.

In some embodiments, the present invention is an in vitro method for typing a sample of a subject suffering from cancer, said method comprising:
a) providing a sample from the subject;
b) quantifying the expression level of c-MAF in said sample using a binding member (e.g. an antibody), variant or fragment as described herein; and c) c-MAF quantified Typing the sample by comparing the expression level of the sample to a predetermined reference level of c-MAF expression;
Wherein the typing step is directed to an in vitro method that provides prognostic information regarding the risk of bone metastasis in the subject.

  In another aspect, the invention is an in vitro method for determining the risk of metastasis in a subject suffering from cancer, using a binding member as described herein The present invention relates to an in vitro method comprising the step of determining the expression level of c-MAF gene in a sample of said subject.

  In a preferred embodiment, the metastasis is a bone metastasis. In one embodiment, expression levels of said genes above mean value + 1 standard deviation indicate an increased risk of early bone metastasis.

  In a preferred embodiment, the bone metastasis is a very early bone metastasis.

  In a preferred embodiment, the bone metastasis is osteolytic metastasis.

  As used herein, "early bone metastasis" relates to bone metastasis that appears 5 years before surgery in patients with breast cancer.

  As used herein, "very early bone metastases" relates to bone metastases that appear 3 years before surgery in patients with breast cancer.

  In some embodiments, the present invention is a method for preventing, inhibiting or reducing the risk of metastasis in a subject suffering from cancer, as described herein A method comprising administering to the subject a binding member (eg, an antibody), variant or fragment thereof. In some embodiments, the metastasis is a bone metastasis.

  In some embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, prostate cancer, and renal cell cancer. In some embodiments, the breast cancer is selected from HER2 + breast cancer, ER + breast cancer, and triple negative breast cancer.

  Thus, the present invention provides methods of treating or diagnosing c-MAF related disorders. In some embodiments, the invention is a method of treating a c-MAF related disorder, comprising an effective amount of one or more binding members (eg, antibodies) of the invention alone or in the art Provided is a method comprising administering to a subject in need thereof in combination with a treatment regimen using another suitable medicament known in the art or described herein.

As is known in the art, the treatment to be administered to a subject suffering from cancer is whether the latter is a malignancy, ie whether it has a high probability of developing metastasis, or It depends on whether the latter is a benign tumor. In the first hypothesis, the generally preferred treatment is a systemic treatment such as chemotherapy, and in the second hypothesis the generally preferred treatment is a local treatment such as radiation therapy.

  Therefore, as described in the present invention, considering that overexpression of c-MAF gene in cancer cells is related to the presence of metastasis (eg, bone metastasis), the expression level of c-MAF gene is It is useful to make decisions regarding the most appropriate treatment for a subject suffering from cancer.

  In certain embodiments, the metastasis is a bone metastasis. In some embodiments, the bone metastasis is osteolytic metastasis.

  In one embodiment, the present invention comprises, in a first step, quantifying c-MAF gene expression levels in a sample of a subject suffering from cancer. In a preferred embodiment, the sample is a tumor tissue sample.

  In another specific embodiment, the method comprises the step of quantifying only c-MAF gene expression level as a single marker, ie the method does not comprise the step of determining the expression level of any additional marker .

  In certain embodiments, the sample may be a primary tumor tissue sample of a subject.

  In one embodiment, the c-MAF gene expression level obtained in a subject's tumor sample is compared to a reference value. In a preferred embodiment, the reference value is c-MAF gene expression of the gene in a control sample. The determination of c-MAF gene expression levels should be related to the values of control or reference samples. Depending on the type of tumor to be analyzed, the exact nature of the control sample may vary. Thus, preferably, the reference sample is c-MAF gene expression measured in a tumor tissue collection of a sample of a subject having a non-metastatic cancer or a biopsy sample of a subject having a non-metastatic cancer It is a sample corresponding to the median of the level.

  Once the c-MAF gene expression level in the sample is measured using the binding member disclosed herein and compared to a reference value, the expression level of the gene is compared to the reference value. If elevated, the subject aims to prevent (if the subject has not yet metastasized) and / or treat metastasis, or to prevent and / or treat metastasis (subject has already metastasized) It can be concluded that it is possible to not seek, to receive treatment, or to receive treatment if it is.

  When primary cancers with or without metastasis have been detected or metastasized, systemic treatments including but not limited to chemotherapy, hormone treatment, immunotherapy or combinations thereof are used . Additionally, radiation therapy and / or surgery may be used. The choice of treatment usually depends on the type of primary cancer, size, location of metastasis, age of the patient, general health status, and type of treatment previously used.

Systemic treatment is treatment that reaches the entire body and prevents or inhibits metastasis (if the subject has not yet developed metastasis) and / or treatment of metastasis (if the subject has already undergone metastasis). It may correspond to a targeted therapy therapy, for example:
Chemotherapy is the use of drugs that destroy cancer cells. Those drugs are usually administered by oral or intravenous route. Occasionally, chemotherapy is used in conjunction with radiation treatment. Preferred chemotherapy treatments for breast cancer include anthracyclines (doxorubicin, epirubicin, pegylated liposomal doxorubicin), taxanes (paclitaxel, docetaxel, albumin nanoparticle conjugated paclitaxel), 5-fluorouracil (5-FU continuous infusion, capecitabine), Vinca alkaloids (Vinorelbine, vinblastine), Gemcitabine, Platinum salts (Cisplatin, Carboplatin), Cyclophosphamide, Etoposide, and combinations of one or more of the above, for example cyclophosphamide / anthracycline +/- 5-fluorouracil Regimens (eg, doxorubicin / cyclophosphamide (AC), epirubicin / cyclophosphamide, (EC) cyclophosphamide / epirubicin / 5-fluoro Lacyl (CEF), cyclophosphamide / doxorubicin / 5-fluorouracil (CAF), 5-fluorouracil / epirubicin / cyclophosphamide (FEC), cyclophosphamide / methotrexate (metothrexate) / 5-fluorouracil (CMF) Anthracyclines / taxanes (eg, doxorubicin / paclitaxel or doxorubicin / docetaxel), docetaxel / capecitabine, gemcitabine / paclitaxel, taxane / platinum regimens (eg, paclitaxel / carboplatin or docetaxel / carboplatin), but are not limited thereto.
Immunotherapy is a treatment that helps the patient's immune system itself to fight cancer. There are several types of immunotherapy used to treat metastases in patients. These include, but are not limited to, cytokines, monoclonal antibodies and anti-tumor vaccines.

  In another embodiment, the treatment is alpha ladin (radiium dichloride-223). Alphaladin uses alpha rays from the decay of radium-223 to kill cancer cells. Radium-223 naturally targets itself for bone metastases thanks to its properties as a calcimimetic. Because alpha radiation has a very short range of 2 to 10 cells (compared to current radiation treatments based on beta or gamma radiation), it causes less damage to surrounding healthy tissues (especially bone marrow). Having similar properties to calcium, radium-223 is found in locations where calcium is used to build bones in the body (as found in skeletal metastases of men with castration resistant advanced prostate cancer (Including the site of abnormal bone growth). Radium-223 is carried by the bloodstream to the site of abnormal bone growth after injection. The location where cancer began to occur in the body is known as the primary tumor. Some of these cells can break off and be transported by blood flow to other parts of the body. Those cancer cells can then colonize at that location of the body to form a new tumor. When this occurs it is called a secondary cancer or metastasis. Most patients with end-stage prostate cancer inflict the greatest burden of the disease on bone. The purpose of using radium-223 is to selectively target this secondary cancer. Any radium-223 not taken up into the bone is quickly routed to the intestine and excreted.

  In another embodiment, the treatment is an mTor inhibitor. In some embodiments, the mTor inhibitor is a dual inhibitor of mTor / PI3 kinase. In some embodiments, mTor inhibitors are used to prevent or inhibit metastasis. In some embodiments, the mTor inhibitor is: ABI 009 (sirolimus), rapamycin (sirolimus), Abraxane (paclitaxel), Absorb (everolimus), Afinitor (everolimus), Afinitor in combination with Gleevec, AS703026 (pimasertib (pimasertib)), Axxess (Umirimus), AZD2014, BEZ235, Biofreedom (Umirimus), BioMatrix (Umirimus), BioMatrix flex (Umirimus), CC115, CC223, Combo Bio-engineered Sirolimus Eluting Stent ORBUS NEICH (Sirolimus), Curaxin CBLC 102 (mepacrine), DE 109 (sirolimus), DS 3078, Endeavor DES (zotarolimus), Endeavor Resolute (zotarolimus), Femara (retrozole), Hocena (anthroquinonol), INK 128, Inspiron (sirolimus), IPI 504 (IR Lettuce pimycin (retaspimycin hydrochloride), KRN 951 (tivozanib), ME 344, MGA 031 (teprizumab), MiStent SES (sirolimus), MKC1, Nobori (umimorimus), OSI 027, OVI 123 (cordycepin), Palomid 529 , PF04691502, Promu Element (everolimus), PWT 33 597, Rapamune (sirolimus), Resolute DES (zotarolimus), RG7422, SAR245409, SF 1126, SGN 75 (borsetuzumab mahodotin (vorsetuzumab mafodotin)), Synergy (everolimus), Taltorvic (ridaforolimus ( ridaforolimus), Tarceva (erlotinib), Torisel (temsirolimus), Xience Prime (everolimus), Xience V (everolimus), Zomaxx (zotarolimus), Zortress (everolimus), zotarolimus-eluting peripheral stent (Peripheral Stent) MEDTRONIC (zotarolimus) , AP23841, AP24170, ARmTOR26, BN107, BN108, Canstatin GENZYME (canstatin), CU906, EC0371, EC0565, KI1004, LOR220, NV128, Rapamycin ONCOIMMUNE (sirolimus), SB2602, Sirolimus PNP SAMYANG BIOPHPMUTILIL TIL , VLI27, VS5584, WYE125132, XL388, Advacan (everolimus), AZD 8055, Cypher Select Plus Sirolimus-eluting coronary stent (sirolimus), Cypher Sirolimus-eluting coronary stent (sirolimus), drug coat Balloon (sirolimus), E-Magic Plus (sirolimus), Emtor (sirolimus), Esprit (everolimus), Evertor (everolimus), HBF0079, LCP-Siro (sirolimus), Limus CLARIS (sirolimus), mTOR inhibitor CELLZOME, Nevo sirolimus-eluting coronary artery stent (sirolimus), nPT-mTOR, Rapacan (sirolimus), Renacept (sirolimus), ReZolve (sirolimus), Rocas (sirolimus), SF1126, Sirolim (sirolimus), Sirolimus NORTH CHINA (sirolimus), Sirolimus RANBAXY (Sirolimus), Sirolimus WATSON Limus) Siropan (sirolimus), Sirova (sirolimus), Supralimus (sirolimus), Supralimus-Core (sirolimus), Tacrolimus WATSON (tacrolimus), TAFA 93, Temsirolimus ACCORD (temsirolimus), Temsirolimus SANDOZ (temsirolimus), TOP 216, Xiience Prime (eberolimus) And Xience V (everolimus). In a specific embodiment, the mTor inhibitor is Afinitor (everolimus) (https://www.afinitor.com/index.jsp?usertrack.filter_applied=true&NovaId=4029462064338207963; last access date November 28, 2012). In another embodiment, everolimus is combined with an aromatase inhibitor (eg, Baselga, J. et al., Everolimus in Postmenopausal Hormone-Receptor Positive Advanced Breast Cancer. 2012. N. Engl. J. Med. 366 (6): 520 -529, which is incorporated herein by reference). In another embodiment, mTor inhibitors may be identified by methods known in the art (eg, Zhou, H. et al. Updates of mTor inhibitors. 2010. Anticancer Agents Med. Chem. 10 (7): 571-81 ( (Which is incorporated herein by reference)). In some embodiments, mTor inhibitors are used to treat or prevent or inhibit metastasis in patients positive for hormone receptors (eg, Baselga, J., el al., Everolimus in Postmenopausal Hormon-Receptor Positive Advanced Breast Cancer. 2012. N. Engl. J. Med. 366 (6): 520-529). In some embodiments, the patient is ER +. In some embodiments, mTor inhibitors are used to treat or prevent or inhibit metastasis in patients with advanced breast cancer. In some embodiments, mTor inhibitors are used in combination with a second treatment. In some embodiments, the second treatment is any treatment described herein.

  In another embodiment, the treatment is a Src kinase inhibitor. In some embodiments, Src inhibitors are used to prevent or inhibit metastasis. In some embodiments, Src kinase inhibitors are selected from the following groups: AZD0530 (salacatinib (saracatinib)), Bosulif (bosutinib (bosutinib)), ENMD 981693, KD 020, KX01, Sprycel (dasatinib (dasatinib)), Yervoy (ipilimumab (ipilimumab (ipilimumab)) A) AP23464, AP23485, AP23588, AZD0424, c-Src kinase inhibitor KISSEI, CU201, KX2361, SKS 927, SRN 004, SUNK 706, TG100435, TG100948, AP23451, Dasatinib HETERO (Dasatinib), Dasatinib VALEANT (Dasatinib), Fontrax) Dasatinib), Src kinase inhibitors KINEX, VX680 (Tozaseruchibu (Tozasertib) lactate), it is selected from XL228 and SUNK706. In some embodiments, the Src kinase inhibitor is dasatinib. In another embodiment, Src kinase inhibitors can be identified by methods known in the art (see, eg, Sen, B. and Johnson, FM. Regulation of Src Family Kinases in Human Cancers. 2011. J. Signal Transduction. 2011: 14 pages (see which is incorporated herein by reference). In some embodiments, Src kinase inhibitors are used to treat or prevent or inhibit metastasis in patients who are positive for SRC responsive signature (SRS). In some embodiments, the patient is SRS + and ER- (eg, Zhang, CH. -F, et al., Latent Bone Metastasis in Breast Cancer Tied to Src-Dependent survival signals. 2009. Cancer Cell. 16: 67- 78, which is incorporated herein by reference). In some embodiments, Src kinase inhibitors are used to treat or prevent or inhibit metastasis in patients with advanced breast cancer. In some embodiments, a Src kinase inhibitor is used in combination with a second treatment. In some embodiments, the second treatment is any treatment described herein.

In another aspect, the treatment is a COX-2 inhibitor. In some embodiments, COX-2 inhibitors are used to prevent or inhibit metastasis. In some embodiments, the COX-2 inhibitors are in the following groups: ABT 963, Acetaminophen ER JOHNSON (acetaminophen), Acular X (ketorolac tromethamine), BAY 1019036 (aspirin), BAY 987111 (diphenhydramine, naproxen sodium), BAYl 1902 (Piroxicam), BCIBUCH 001 (Ibuprofen), Capoxigem (apricoxib), CS 502, CS 670 (Perbiprofen (pelubiprofen)), Diclofenac HPBCD (Diclofenac), Diactin (Ketoprofen), GW406381, HCT1026 (Nitroflurbiprofen (Nitroflurpr ofen), Hyalargese-D (Diclofenac), HydrocoDex (Acetaminophen, Dextromethorphan, Hydrocodone), Ibuprofen Sodium PFIZER (Ibuprofen Sodium), Ibuprofen with Acetaminophen PFIZER (Acetaminophen, Ibuprofen), Impracor (Ketoprofen), IP 880 (Diclofenac), IP 940 (Indometacin), ISV 205 (Diclofenac Sodium), JNS013 (Acetaminophen, Tramadol Hydrochloride), Ketoprofen TDS (Ketoprofen), LTNS 001 (Naproxene etemesil), Mesalamine SAL X (mesalamine), Mesalamine SOFAR (mesalamine), Mesalazine (mesalamine), ML3000 (licofelone (licofelone)),
MRX 7 EAT (etodolac), Naproxen IROKO (naproxen), NCX 4016 (nitroaspirin), NCX 701 (nitro acetaminophen), Nuprin SCOLR (ibuprofen), OMS 103 HP (amitryptiline hydrochloride, ketoprofen, oxymetazoline hydrochloride), Oralease (diclofenac) , OxycoDex (dextromethorphan, oxycodone), P54, PercoDex (acetaminophen, dextromethorphan, oxycodone), PL3100 (naproxen, phosphatidylcholine), PSD 508, R-Ketoprofen (ketoprofen), Remura (bromfenac sodium), ROX 828 (Ketorolac tromethamine), RP 19583 Ketoprofen lysine), RQ00317076, SDX101 (R-etodolac), TDS943 (diclofenac sodium), TDT 070 (ketoprofen), TPR100, TQ1011 (ketoprofen), TT063 (S-flurbiprofen), UR 8880 (simicoxib (cimicoxib)), V0498TA01A (Ibuprofen), VT122 (etodolac, propranolol), XP20B (acetaminophen, dextropropoxyphene), XP21 B (diclofenac potassium), XP21 L (diclofenac potassium), Zoenasa (acetylcysteine, mesalamine), Acephen, Actifed Plus, Actifed- P, Acular, Acular LS, Acular PF, Acular X, Acuvail, Advil, Advil Allergy Sinus, Advil Cold and Sinus, Advil Congestion Relief, Advil PM, Advil PM Capsule, Air Salonpas, Airtal, Alcohol-Free NyQuil Cold & Flu Relief, Aleve, Aleve ABDI IBRAHIM, Aleve -D, Alka-Seltzer, Alka-Seltzer BAYER, Alka-Seltzer Extra Strength, Alka-Seltzer Lemon-Lime, Alka-Seltzer Original, Alka-Seltzer Plus, Alka-Seltzer plus C Alka-Seltzer plus Cold and Cough Formula, Alka-Seltzer Plus Day and Night Cold Formula, Alka-Seltzer Plus Day Non-Drowsy Cold Formula, Alka-Seltzer Plus Flu Formula, Alka-Seltzer Plus Night Cold Formula, Alka -Seltzer Plus Sinus Formula, Alka-Seltzer Plus Sparkling Original Cold Formula, Alka-Seltzer PM, Alka-Seltzer Wake-Up Call, Anacin, Anaprox, Anapr x MINERVA, Ansaid, Apitoxin, Apranax, Apranax abdi, Arcoxia, Arthritis Formula Bengay, Arthrotec, Asacol, Asacol HD, Asacol MEDUNA ARZNEIMITTEL, Asacol ORIFARM, Aspirin BAYER, Aspirin Complex, Aspirin BAY 987111, BAYl 1902, BCIBUCH 001, Benadryl Allergy, Benadryl Day and Night, Benylin 4 Flu, Benylin Cold and Flu, Benylin Cold and Flu Day and Night, Benylin Cold and Sinus Day, Benylin Cold and Sinus Plus, Benylin Day and Night Cold and Flu Relief, Benylin 1 All-In-One, Brexin, Brexin ANGELINI, Bromday, Bufferin, Buscopan Plus, Caldolor , Calmatel, Cambia, Canasa, Capoxigem, Cataflam, Celebrex, Celebrex ORIFARM, Children's Advil Allergy Sinus, Children's Tylenol, Children's Tylenol Co gh and Runny Nose, Children's Tylenol plus cold, Children's Tylenol plus Cold and Cough, Children's Tylenol plus cold and stuffy nose, Children's Tylenol plus Flu, Children's Tylenol plus cold & allergy, Children's Tylenol plus Cough & Runny Nose, Children's Tylenol plus Cough & Sore Throat, Children's Tylenol plus multi symptom cold, Clinoril, Codral Cold and Flu, Codral Day a d Night Day Tablets, Codral Day and Night Night Tablets, Codral Nightime, Colazal, Combanox, Contac Cold plus Flu, Contac Cold plus Flu Non-Drowsy, Coricidin D, Coricidin HBP Cold and Flu, Coricidin HBP Day and Night Multi-Symptom Cold Coricidin HBP Maximum Strength Flu, Coricidin HBP Nighttime Multi-Symptom Cold, Coricidin II Extra Strength Cold and Flu,
CS502, CS670, Daypro, Daypro, Daypro, DDS06C, Demazin Cold and Flu, Demazin Cough, Cold and Flu, Demazin day / night Cold and Flu, Demazin PE Cold and Flu, Demazin PE day / night Cold and Flu, Diclofenac HPBCD, Dimetappapp Day Relief, Dimetapp Multi-Symptom Cold and Flu, Dimetapp Night Relief, Dimetapp Pain and Fever Relief, Dimetapp PE Sinus Pain, Dimetapp PE Sinus Pain plus Allergy, ipentum, Diractin, Disprin Cold 'n' Fever, Disprin Extra, Disprin Forte. Disprin Plus, Dristan Cold, Dristan Junior, Drixoral Plus, Duexis, Dynastat, Efferalgan, Efferalgan Plus Vitamin C, Efferalgan Vitamin C, Elixsure IB, Excedrin Back and Body, Excedrin Migraine, Excedrin PM, Excedrin Sl , Fansamac, Feldene, FeverAll, Fiorinal, Fiorinal with Codeine, Flanax, Flector Patch, Flucam, Fortagesic, G erbin, Giazo, Gladio, Goody's Back Pain, Goody's Cool Orange, Goody's Extra Strength, Goody's PM, Greaseless Bengay, GW406381, HCT1026, He Xing Yi, Hyanalgese-D, HydrocoDex, Ibuprofen Sodium PFIZER, Ibuprofen, Acetaminophen PFIZER, Icy Hot SANOFI AVENTIS, Impracor, Indocin, Indomethacin APP PHARMA, Indomethacin MYLAN, Infants' Tylenol, IP880, IP940, Iremod, ISV 205, JNS013, Jr. Tylenol, Junifen, Junior Strength Advil, Junior Strength Motrin, Ketoprofen TDS, Lemsip Max, Lemsip Max All in One, Lemsip Max All Night, Lemsip Max Cold and Flu, Lialda, Listerine Mouth Wash, Lloyds Cream, Lodine, Lorfit P, Loxonin , LTNS 001, Mersyndol, Mesalamine SALIX, Mesalamine SOFAR, Mesalazine, Mesasal GLAXO, Mesasal SANOFI, Mesalid, Metsal Heat Rub, Midol Complete, Mi dol Extended Relief, Midol Liquid Gels, Midol PM, Midol Teen Formula, Migranin COATED TABLETS, ML 3000, Mobic, Mohrus, Motrin, Motrin Cold and Sinus Pain, Motrin PM, Movalis ASPEN, MRX7 EAT, Nalfon, Nalfon PEDINOL Napresyn, Naprosyn RPG LIFE SCIENCE, Naproxen IROKO, NCX 4016, NCX 701, NeoProfen LUNDBECK, Nevanac, Nexcede, Niflan, Norgesic MEDICIS, Novalgin, Nuprin S OLR, Nurofen, Nurofen Cold and Flu, Nurofen Max Strength Migraine, Nurofen Plus,
Nuromol, NyQuil with Vitamin C, Ocufen, OMS 103 HP, Oralease, Orudis ABBOTT JAPAN, Oruvail, Ostleuc, OxycoDex, P54, Panadol, Panadol, Panadol Actifast, Paradine, Paraxa, Parfenac, Pedena, Pennsaid, Pentasa, Pentasa, Pentasa ORP Demi, PercoDex, Percogesic, Perfalgan, PL2200, Ponstel, Prexige, Prolensa, PSD 508, R-Ketoprofen, Rantudil, Relafen, Remura, Rob axisal, Rotec, Rowasa, ROX 828, RP 19583, RQ00317076, Rubor, Salofalk, Salonpas, Saridon, SDX 101, Seltouch, sfRowasa, Shinbaro, Sinumax, Sinutab, Sinutab, sinus, Spalt, Sprix, Strefen, Sudafed Cold Sic and Sinus, Sudafed PE Cold plus Cough, Sudafed PE Pressure plus Pain, Sudafed PE, Severe Cold, Sudafed PE Sinus Day plus Night Relief Day Tablets, Sudafe d PE Sinus Day plus Night Relief Night Tablets, Sudafed PE Sinus plus Anti-inflammatory Pain Relief,
Sudafed Sinus Advance, Surgam, Synalgos-DC, Synflex, Tavist energy / sinus / headache, TDS 943, TDT 070, Theraflu Cold and Sore Throat, Theraflu Daytime Severe Cold and Cough, Theraflu Daytime Warming Relief, Theraflu Warming Rem. , Theraflu Warming Relief Cold and Chest Congestion, Thomapyrin, Thomapyrin C, Thomapyrin Effervescent, T omapyrin Medium, Tilcotil, Tispol, Tolectol, Toradol, TPR100, TQ1011, Trauma-Salbe, Trauma-Salbe Kwizda, Treo, Treximet, Trovex, TT063, Tylenol, Tylenol Allergy Multi-Symptom, Tylitolitole, Tylenol Cold & Cough Nighttime, Tylenol Cold and Sinus Daytime, Tylenol Cold and Sinus Nighttime, Tylenol Cold Head Congestion Severe, Tylenol Cold MultiSymptom Daytime, Tylenol Cold MultiSymptom Nighttime Liquid, Tylenol Cold MultiSymptom Severe, Tynolol Non-Drowsiness Formula, Tynolol Cold Severe Congestion Daytime, Tynolol Complete Cold, Cough and Flu Night time, Tynolol Tylenol Sinus Congestion & Pain Daytime, Tylenol Sinus Congestion & Pain Nighttime, Tyl nol Sinus Congestion & Pain Severe, Tylenol Sinus Severe Congestion Daytime, Tylenol Ultra Relief, Tylenol with Caffeine and Codeine phosphate, Tylenol with Codeine phosphate, Ultra Strength Bengay Cream, Ultracet, UR 8880, V 0 498 , Voltaren Emulgel, Voltaren GEL, Voltaren NOVARTIS CONSUMER HEALTH GMBH, Voltaren R, VT122, Xefo, Xefo Rapid, Xefocam, Xibrom, XL3, Xodol, XP20B, XP21B, XP21L, selected from Zipsor and Zoenasa. In another embodiment, COX-2 inhibitors can be identified by methods known in the art (see, eg, Dannhardt, G. and Kiefer, W. Cyclooxygenase inhibitors-current status and future prospects. 2001. Eur. J. Med. Chem. 36: 109-126 (which is incorporated herein by reference). In some embodiments, a COX-2 inhibitor is used to treat or prevent or inhibit metastasis in a patient with advanced breast cancer. In some embodiments, a COX-2 inhibitor is used in combination with a second treatment. In some embodiments, the second treatment is any treatment described herein. In some embodiments, the COX-2 inhibitor is Denosumab, Zometa (https://www.us.zometa.com/index.jsp?usertrack.filter_applied=true&NovaId=2935376934467633633; Last Access Date December 2, 2012) , Carbozantinib or Cabozantinib, an antibody or peptide that blocks PTH LH (parathyroid hormone-like hormone) or PTHrP (parathyroid hormone related protein), and is used in combination with a second treatment selected from the group consisting of everolimus.

In another aspect, treatment agents used to avoid and / or prevent bone degradation include, but are not limited to:
• Parathyroid hormone (PTH) inhibitors and parathyroid hormone (PTHLH) inhibitors (including blocking antibodies) or their recombinants (teriparatide corresponding to amino acids 7 to 34 of PTH). This hormone acts by stimulating osteoclasts to enhance their activity.
Strontium ranelate is an alternative oral treatment, which stimulates osteoblast proliferation and inhibits osteoclast proliferation, so it is called "dual action bone agents" (DABA) Form part of a group of drugs.
"Estrogen receptor modulator" (SERM) refers to compounds that interfere with or inhibit estrogen binding to the receptor regardless of mechanism. Examples of estrogen receptor modulators include estrogen progestagen, estradiol, droloxifene, raloxifene, lasofoxifene, TSE-424, tamoxifen, idoxifene, LY353381, LY117081, toremifene, fulvestrant (especially) fluvestrant), 4- [7- (2,2-dimethyl-1-oxopropoxy-4-methyl-2- [4- [2- (1-piperidinyl) ethoxy] phenyl] -2H-1-benzopyran-3- Yl] -phenyl-2,2-dimethylpropanoate 4,4'-dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone and SH646.
Calcitonin directly inhibits osteoclast activity via the calcitonin receptor. Calcitonin receptors have been identified on the surface of osteoclasts.
-Bisphosphonates prevent and treat diseases with bone resorption and resorption, such as osteoporosis and cancer with bone metastases (the latter with or without hypercalcemia associated with breast and prostate cancer) Is a group of medicines used for Examples of bisphosphonates that can be used in the therapy designed according to the fifth method of the present invention include nitrogen-containing bisphosphonates (eg pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, Examples include, but are not limited to, incadronate, zoledronate or zoledronic acid and non-nitrogen containing bisphosphonates such as etidronate, clodronate, tiludronate and the like.
"Cathepsin K inhibitors" refer to compounds that interfere with cathepsin K cysteine protease activity. Non-limiting examples of cathepsin K inhibitors include 4-amino-pyrimidine-2-carbonitrile derivatives (described in International Patent Application Publication WO 03/020278 under the name of Novartis Pharma GMBH), publication WO 03/020721. Novartis Pharma GMBH) and the pyrrolo-pyrimidines described in the publication WO 04/000843 (ASTRAZENECA AB), as well as the publication PCT WO 00/55126 of Axys Pharmaceuticals, Merck Frasst Canada & Co. And inhibitors described in WO 01/49288 of Axys Pharmaceuticals.
-"DKK-1 (Dickkopf-1) inhibitor" as used herein refers to any compound capable of reducing DKK-1 activity. DKK-1 is a soluble Wnt pathway antagonist that is mainly expressed in adult bone and upregulated in myeloma patients with osteolytic lesions. Agents that target DKK-1 may play a role in the prevention of osteolytic bone disease in multiple myeloma patients. Novartis BHQ 880 is a first in class fully human anti-DKK-1 neutralizing antibody. Preclinical studies support the hypothesis that BHQ 880 promotes bone formation, thereby inhibiting tumor-induced osteolytic disease (Ettenberg S. et al. American Association for Cancer Research Annual Meeting. April 12 -16, 2008; San Diego, Calif. Abstract).
"Dual inhibitors of MET and VEGFR2" as used herein are potent dual inhibitors of the MET and VEGF pathways, designed to block MET-driven tumor escape It refers to any compound. MET is expressed not only in tumor cells and endothelial cells, but also in osteoblasts (cells that form bone) and osteoclasts (cells that remove bone). HGF binds to MET in all of these cell types, giving the MET pathway an important role in multiple autocrine and paracrine loops. Activation of MET in tumor cells appears to be important in establishing metastatic bone lesions. At the same time, activation of the MET pathway in osteoblasts and osteoclasts is a pathological feature of bone metastasis, including abnormal bone growth (ie blastic lesions) or destruction (ie lytic lesions) Can bring Thus, targeting of the MET pathway may be a viable strategy to prevent the establishment and progression of metastatic bone lesions. Cabozantinib (Exelixis, Inc), formerly known as XL184 (CAS 849217-68-1), is designed to block MET-driven tumor escape and is potent for the MET and VEGF pathways It is a dual inhibitor. In multiple preclinical studies, cabozantinib has been shown to kill tumor cells, reduce metastasis, and inhibit angiogenesis (the formation of new blood vessels necessary to support tumor growth). Another preferred dual inhibitor is E7050 (N- [2-fluoro-4-({2- [4- (4-methylpiperazin-1-yl) piperidin-1-yl] carbonylaminopyridin-4-yl } Oxy) phenyl] -N '-(4-fluorophenyl) cyclopropane-1,1-dicarboxamide (2R, 3R) -tartrate) (CAS 928037-13-2) or foretinib (foretinib) (GSK 1363089, XL 880, CAS 849217-64-7).
-"RANKL inhibitor" as used herein refers to any compound capable of reducing RANK activity. RANKL is found on the surface of the osteoblastic membrane of stromal cells and T-lymphocytes, and these T-lymphocytes are the only cells that have demonstrated their ability to secrete it. Its main function is the activation of osteoclasts, the cells involved in bone resorption. RANKL inhibitors by blocking RANKL binding to its receptor (RANK), blocking RANK-mediated signaling, or reducing RANKL expression by blocking RANKL transcription or translation It can work. RANKL antagonists or inhibitors suitable for use in the present invention include, but are not limited to:
Preferred RANK proteins, which can bind to RANKL and comprise all or a fragment of the extracellular domain of the RANK protein. Soluble RANK may comprise the signal peptide and extracellular domain of mouse or human RANK polypeptide, or alternatively, the mature form of the protein from which the signal peptide has been removed may be used.
• Osteoprotegerin or a variant thereof having RANKL binding ability.
RANKL specific antisense molecules.
Ribozymes capable of processing RANKL transcripts.
Specific anti-RANKL antibodies. An "anti-RANKL antibody or an antibody directed against RANKL" is any antibody capable of specifically binding to a ligand (RANKL) of an activating receptor for nuclear factor BB, which inhibits one or more functions of RANKL. And as understood herein. The antibodies can be prepared using any method known to one skilled in the art. Thus, polyclonal antibodies are prepared by immunizing animals with the protein to be inhibited. Monoclonal antibodies are prepared using the method described by Kohler, Milstein et al. (Nature, 1975, 256: 495). Preferred antibodies in the context of the present invention include intact antibodies comprising variable antigen binding and constant regions, fragments "Fab", "F (ab ') 2" and "Fab", Fv, scFv, diabodies and Bispecific antibodies are included.
Specific anti-RANKL Nanobodies. Nanobodies are antibody-derived therapeutic proteins that include the unique structural and functional properties of naturally occurring heavy chain antibodies. Nanobody technology was first developed after the discovery that camelids (camels and llamas) have fully functional antibodies lacking light chains. The general structure of a nanobody is
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
Here, FR1 to FR4 are framework regions 1 to 4, and CDR1 to CDR3 are complementarity determining regions 1 to 3. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Importantly, the cloned and isolated VHH domain is a completely stable polypeptide that has the full antigen binding ability of the original heavy chain antibody. These newly discovered VHH domains with unique structural and functional properties form the basis of a new generation of therapeutic antibodies that Ablynx has named Nanobodies.

  In one embodiment, the RANKL inhibitor is selected from the group consisting of a RANKL specific antibody, a RANKL specific Nanobody and osteoprotegerin. In a specific embodiment, the anti-RANKL antibody is a monoclonal antibody. In an even more specific embodiment, the anti-RANKL antibody is denosumab (Pageau, Steven C. (2009). MAbs 1 (3): 210-215, CAS No. 615258-40-7) (all of which The contents of which are hereby incorporated by reference). Denosumab is a fully human monoclonal antibody that binds RANKL and prevents its activation (it does not bind to the RANK receptor). Various embodiments of denosumab are disclosed in U.S. Patent Nos. 6,740,522; 7,411,050; 7,097,834; 7,364,736 (the entire contents of each of these). Is hereby incorporated by reference in its entirety). In another embodiment, the RANKL inhibitor is an antibody, antibody fragment or fusion construct that binds to the same epitope as denosumab.

  In a preferred embodiment, the anti-RANKL Nanobody is any of the Nanobodies as described in WO2008142164, the contents of which are incorporated herein by reference. In an even more preferred embodiment, the anti-RANKL antibody is ALX-0141 (Ablynx). ALX-0141 was designed to inhibit postmenopausal osteoporosis, rheumatoid arthritis, cancer and bone loss associated with certain medications, and to restore the balance of healthy bone metabolism.

  In a preferred embodiment, agents that prevent bone degradation include bisphosphonates, RANKL inhibitors, PTH and PTHLH inhibitors or PRG analogues, strontium lanerate, DKK-1 inhibitors, dual inhibitors of MET and VEGFR2, estrogen receptor modulators, radium- 223 selected from the group consisting of calcitonin and cathepsin K inhibitors. In a more preferred embodiment, the agent that prevents bone degradation is a bisphosphonate. In an even more preferred embodiment, the bisphosphonate is zoledronic acid.

  In one embodiment, a CCR5 antagonist is administered to prevent or inhibit metastasis of a primary breast cancer tumor to bone. In one embodiment, the CCR5 antagonist is a large molecule. In another embodiment, the CCR5 antagonist is a small molecule. In some embodiments, the CCR5 antagonist is maraviroc (Velasco-Velaquez, M. et al., 2012. CCR5 Antagonist Blocks Metastasis of Basal Breast Cancer Cells. Cancer Research. 72: 3839-3850). In some embodiments, the CCR5 antagonist is vicriviroc (Velasco-Vela'quez, M. et al., 2012. CCR5 Antagonist Blocks Metastasis of Basal Breast Cancer Cells. Cancer Research. 72: 3839-3850). In some embodiments, the CCR5 antagonist is aplaviroc (Demarest J. F. et al., 2005. Update on Aplaviroc: An HIV Entry Inhibitor Targeting CCR5. Retrovirology 2 (Suppl. 1): S13). In some embodiments, the CCR5 antagonist is a spiropiperidine CCR5 antagonist (Rotstein DM, et al., 2009. Spiropiperidine CCR5 antagonists. Bioorganic & Medicinal Chemistry Letters. 19 (18): 5401-5406). In some embodiments, the CCR5 antagonist is INCB009471 (Kuritzkes, DR 2009. HIV-1 entry inhibitors: an overview. Curr. Opin. HIV AIDS. 4 (2): 82-7).

  In a preferred embodiment, the dual inhibitor of MET and VEGFR2 is selected from the group consisting of cabozantinib, foretinib and E7050.

  In a preferred embodiment, the radium 223 treatment is alpha ladin.

  Alternatively, combination treatment may be performed in which two or more of the agents mentioned above are combined to treat and / or prevent metastasis, or the agent may be used in other supplements (e.g. It can be combined with calcium or vitamin D) or hormone treatment.

  Once the c-MAF gene expression level in the sample is measured and compared to a reference value (eg, c-MAF gene expression level in a control sample), the expression level of the gene is compared to the reference value. If elevated, this indicates that the subject is capable of receiving treatment aimed at avoiding or preventing bone degradation.

Illustrative examples of agents used to avoid and / or prevent bone degradation include, but are not limited to:
• Parathyroid hormone (PTH) and parathyroid hormone (PTHLH) inhibitors (including block of antibodies) or their recombinants (teriparatide corresponding to amino acids 7 to 34 of PTH). This hormone acts by stimulating osteoblasts to increase their activity.
Strontium ranelate is an alternative oral treatment and stimulates osteoblast proliferation and inhibits osteoclast proliferation, so it is part of a group of drugs called "double acting bone agents" (DABA) Form
"Estrogen receptor modulators" (SERM) refers to compounds that interfere with or inhibit estrogen binding to the receptor regardless of mechanism. Examples of estrogen receptor modulators include estrogen progestagen, estradiol, droloxifene, raloxifene, lasophoxifene, TSE-424, tamoxifen, idoxifene, LY353381, LY117081, toremifene, fulvestrant, 4- [7- 2,2-Dimethyl-1-oxopropoxy-4-methyl-2- [4- [2- (1-piperidinyl) ethoxy] phenyl] -2H-1-benzopyran-3-yl] -phenyl-2,2- Dimethylpropanoate 4,4'-Dihydroxybenzophenone-2,4-dinitrophenyl-hydrazone and SH 646. Calcitonin directly inhibits osteoclast activity via calcitonin receptor. Chromatography has been identified on the surface of osteoclasts.
-Bisphosphonates prevent and treat diseases with bone resorption and resorption, such as osteoporosis and cancer with bone metastases (the latter with or without hypercalcemia associated with breast and prostate cancer) Is a group of medicines used for Examples of bisphosphonates that can be used in the treatment designed by the fifth method of the present invention include nitrogen-containing bisphosphonates (eg, pamidronate, neridronate, olpadronate, alendronate, ibandronate, risedronate, incadronate, zoledronate or Examples include, but are not limited to, zoledronic acid) and non-nitrogen containing bisphosphonates (eg, etidronate, clodronate, tiludronate, etc.).
-Alpha ladin (radiium dichloride-223). Alphaladin uses alpha rays from the decay of radium-223 to kill cancer cells. Radium-223 naturally targets itself for bone metastases thanks to its properties as a calcimimetic. Because alpha rays have a very short range of 2-10 cells (compared to current radiation treatments based on beta or gamma rays), they cause less damage to surrounding healthy tissues (especially bone marrow). Having similar properties to calcium, radium-223 is where calcium is used to build bones in the body (as found in skeletal metastases in men with advanced castration resistant prostate cancer It is drawn to faster abnormal bone growth sites). Radium-223 is carried by the bloodstream to the site of abnormal bone growth after injection. Where cancer has begun to occur in the body is known as the primary tumor. Some of these cells can break off and be transported by blood flow to other parts of the body. Those cancer cells can then colonize that part of the body and form new tumors. When this occurs it is called a secondary cancer or metastasis. Most patients with end-stage prostate cancer inflict the greatest burden of the disease on bone. The purpose of using radium-223 is to selectively target this secondary cancer. Any radium-223 that is not taken up into the bone is promptly sent to the intestine and excreted.
"Cathepsin K inhibitors" refer to compounds that interfere with cathepsin K cysteine protease activity. Non-limiting examples of cathepsin K inhibitors include 4-amino-pyrimidine-2-carbonitrile derivatives (described in International Patent Application Publication WO 03/020278 under the name of Novartis Pharma GMBH), publication WO 03/020721. Novartis Pharma GMBH) and the pyrrolo-pyrimidines described in the publication WO 04/000843 (ASTRAZENECA AB), as well as the publication PCT WO 00/55126 of Axys Pharmaceuticals, Merck Frasst Canada & Co. And inhibitors described in WO 01/49288 of Axys Pharmaceuticals.
-"DKK-1 (Dickkopf-1) inhibitor" as used herein refers to any compound capable of reducing DKK-1 activity. DKK-1 is a soluble Wnt pathway antagonist that is mainly expressed in adult bone and upregulated in myeloma patients with osteolytic lesions. Agents that target DKK-1 may play a role in the prevention of osteolytic bone disease in multiple myeloma patients. Novartis BHQ 880 is a first in class fully human anti-DKK-1 neutralizing antibody. Preclinical studies support the hypothesis that BHQ 880 promotes bone formation, thereby inhibiting tumor-induced osteolytic disease (Ettenberg S. et al. American Association for Cancer Research Annual Meeting. April 12-16, 2008; San Diego, Calif. Abstract).
"Dual inhibitors of MET and VEGFR2" as used herein are potent dual inhibitors of the MET and VEGF pathways, designed to block MET-driven tumor escape It refers to any compound. MET is expressed not only in tumor cells and endothelial cells, but also in osteoblasts (cells that form bone) and osteoclasts (cells that remove bone). HGF binds to MET in all of these cell types, giving the MET pathway an important role in multiple autocrine and paracrine loops. Activation of MET in tumor cells appears to be important in establishing metastatic bone lesions. At the same time, activation of the MET pathway in osteoblasts and osteoclasts is a pathological feature of bone metastasis, including abnormal bone growth (ie blastic lesions) or destruction (ie lytic lesions) Can bring Thus, targeting of the MET pathway may be a viable strategy for the establishment and prevention of metastatic bone lesions. Cabozantinib (Exelixis, Inc), formerly known as XL184 (CAS 849217-68-1), is a potent dual inhibitor of the MET and VEGF pathways, designed to block MET-driven tumor escape It is. In multiple preclinical studies, cabozantinib has been shown to kill tumor cells, reduce metastasis, and inhibit angiogenesis (the formation of new blood vessels necessary to support tumor growth). Another preferred dual inhibitor is E7050 (N- [2-fluoro-4-({2- [4- (4-methylpiperazin-1-yl) piperidin-1-yl] carbonylaminopyridin-4-yl } Oxy) phenyl] -N '-(4-fluorophenyl) cyclopropane-1,1-dicarboxamide (2R, 3R) -tartrate) (CAS 928037-13-2) or foretinib (GSK 1363089, XL 880, CAS 849 217-64) Also known as -7).
-"RANKL inhibitor" as used herein refers to any compound capable of reducing RANK activity. RANKL is found on the surface of the osteoblastic membrane of stromal cells and T-lymphocytes, and these T-lymphocytes are the only cells that have demonstrated their ability to secrete it. Its main function is the activation of osteoclasts, the cells involved in bone resorption. RANKL inhibitors by blocking RANKL binding to its receptor (RANK), blocking RANK-mediated signaling, or reducing RANKL expression by blocking RANKL transcription or translation It can work. RANKL antagonists or inhibitors suitable for use in the present invention include, but are not limited to:
Preferred RANK proteins, which can bind to RANKL and comprise all or a fragment of the extracellular domain of the RANK protein. Soluble RANK may comprise the signal peptide and extracellular domain of mouse or human RANK polypeptide, or alternatively, the mature form of the protein from which the signal peptide has been removed may be used.
• Osteoprotegerin or a variant thereof having RANKL binding ability.
RANKL specific antisense molecules.
Ribozymes capable of processing RANKL transcripts.
Specific anti-RANKL antibodies. An "anti-RANKL antibody or an antibody directed against RANKL" is any antibody capable of specifically binding to a ligand (RANKL) of an activating receptor for nuclear factor BB, which inhibits one or more functions of RANKL. And as understood herein. The antibodies can be prepared using any method known to one skilled in the art. Thus, polyclonal antibodies are prepared by immunizing animals with the protein to be inhibited. Monoclonal antibodies are prepared using the method described by Kohler, Milstein et al. (1975) Nature, 256: 495). Preferred antibodies in the context of the present invention include intact antibodies comprising variable antigen binding and constant regions, fragments "Fab", "F (ab ') 2" and "Fab", Fv, scFv, diabodies and Bispecific antibodies are included.
Specific anti-RANKL Nanobodies. Nanobodies are antibody-derived therapeutic proteins that include the unique structural and functional properties of naturally occurring heavy chain antibodies. Nanobody technology was first developed after the discovery that camelids (camels and llamas) have fully functional antibodies lacking light chains. The general structure of a nanobody is
FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
Here, FR1 to FR4 are framework regions 1 to 4, and CDR1 to CDR3 are complementarity determining regions 1 to 3. These heavy chain antibodies contain a single variable domain (VHH) and two constant domains (CH2 and CH3). Importantly, the cloned and isolated VHH domain is a completely stable polypeptide that has the full antigen binding ability of the original heavy chain antibody. These newly discovered VHH domains with unique structural and functional properties form the basis of a new generation of therapeutic antibodies that Ablynx has named Nanobodies.

  In one embodiment, the RANKL inhibitor is selected from the group consisting of a RANKL specific antibody, a RANKL specific Nanobody and osteoprotegerin. In certain embodiments, the anti-RANKL antibody is a monoclonal antibody. In an even more specific embodiment, the anti-RANKL antibody is denosumab (Pageau, Steven C. (2009) mAbs 1 (3): 210-215, CAS No. 615258-40-7) (this content is hereby incorporated by reference in its entirety). Incorporated herein by reference). Denosumab is a fully human monoclonal antibody that binds to RANKL and prevents its activation (it does not bind to the RANK receptor). Various embodiments of denosumab are disclosed in U.S. Patent Nos. 6,740,522; 7,411,050; 7,097,834; 7,364,736 (the entire contents of each of these). Is hereby incorporated by reference). In another embodiment, the RANKL inhibitor is an antibody, antibody fragment or fusion construct that binds to the same epitope as denosumab.

  In a preferred embodiment, the anti-RANKL Nanobody is any of the Nanobodies as described in WO2008142164, the contents of which are incorporated herein by reference. In an even more preferred embodiment, the anti-RANKL antibody is ALX-0141 (Ablynx). ALX-0141 was designed to inhibit bone loss associated with postmenopausal osteoporosis, rheumatoid arthritis, cancer and certain drugs, and to restore the balance of healthy bone metabolism.

  In a preferred embodiment, agents that prevent bone degradation include bisphosphonates, RANKL inhibitors, PTH and PTHLH inhibitors or PRG analogues, strontium lanerate, DKK-1 inhibitors, dual inhibitors of MET and VEGFR2, estrogen receptor modulators, radium- 223, selected from the group consisting of calcitonin and cathepsin K inhibitors. In a more preferred embodiment, the agent that prevents bone degradation is a bisphosphonate. In an even more preferred embodiment, the bisphosphonate is zoledronic acid.

  In one embodiment, a CCR5 antagonist is administered to prevent or inhibit metastasis of a primary breast cancer tumor to bone. In one embodiment, the CCR5 antagonist is a large molecule. In another embodiment, the CCR5 antagonist is a small molecule. In some embodiments, the CCR5 antagonist is Maraviroc (Velasco-Velaquez, M. et al., 2012. CCR5 Antagonist Blocks Metastasis of Basal Breast Cancer Cells. Cancer Research. 72: 3839-3850). In some embodiments, the CCR5 antagonist is Vicriviroc (Velasco-Velaquez, M. et al., 2012. CCR5 Antagonist Blocks Metastasis of Basal Breast Cancer Cells. Cancer Research. 72: 3839-3850). In some embodiments, the CCR5 antagonist is Aplaviroc (Demarest J. F. et al., 2005. Update on Aplaviroc: An HIV Entry Inhibitor Targeting CCR5. Retrovirology 2 (Suppl. 1): S13). In some embodiments, the CCR5 antagonist is a spiropiperidine CCR5 antagonist (Rotstein DM, et al., 2009. Spiropiperidine CCR5 antagonists. Bioorganic & Medicinal Chemistry Letters. 19 (18): 5401-5406). In some embodiments, the CCR5 antagonist is INCB009471 (Kuritzkes, DR 2009. HIV-1 entry inhibitors: an overview. Curr. Opin. HIV AIDS. 4 (2): 82-7).

  In a preferred embodiment, the dual inhibitor of MET and VEGFR2 is selected from the group consisting of cabozantinib, foretinib and E7050.

  In a preferred embodiment, the radium 223 treatment is alpha ladin.

  A further aspect of the present invention is the use of isolated isolated, for example for inhibiting transcription and / or translation of the nucleic acid encoding said c-MAF, the expression of which is to be inhibited, eg the activity of c-MAF is to be inhibited. Use of "antisense" nucleic acids. Antisense nucleic acids are potential targets for drugs, either by conventional base complementarity or when bound to double stranded DNA, for example, through specific interactions in the large groove of the double helix. It can be combined. Generally, these methods refer to a series of techniques commonly used in the art, including any method based on specific binding to oligonucleotide sequences.

  Small interfering RNAs or siRNAs are agents that can inhibit target gene expression by RNA interference. The siRNA can be chemically synthesized, obtained by in vitro transcription, or synthesized in vivo in target cells. Typically, the siRNA consists of double stranded RNA 15 to 40 nucleotides in length, and may include 1 to 6 nucleotides of 3 'and / or 5' overhangs. The length of the overhanging region is independent of the total length of the siRNA molecule. siRNAs act by degradation or silencing of target messengers following transcription.

The siRNA of the present invention is substantially homologous to the mRNA of the gene encoding c-MAF or the gene sequence encoding the above protein. "Substantially homologous" is understood to have a sequence that is sufficiently complementary or similar to the target mRNA so that the siRNA can degrade the latter by RNA interference. Suitable siRNAs for causing the interference include siRNAs formed by RNA, as well as siRNAs including various chemical modifications, for example:
· SiRNAs in which the linkage between nucleotides differs from the linkage found in nature (eg, phosphorothioate linkage)
Conjugates of functional reagents such as fluorophores with RNA chains Modification of the ends of RNA chains, in particular at the 3 'ends, by modification with various hydroxyl functional groups at the 2' position 2'-O-methyl ribose Or nucleotides with modified sugars such as O-alkylated residues at the 2 'position such as 2'-O-fluororibose, halogenated bases such as 5-bromouracil and 5-iodouracil, Nucleotides having modified bases such as alkylated bases (e.g. 7-methyl guanosine) are included.

  On the other hand, the present invention also contemplates the use of DNA enzymes which inhibit the expression of the c-MAF gene of the present invention. DNA enzymes incorporate some of the mechanistic features of both antisense and ribozyme technologies. DNA enzymes recognize specific target nucleic acid sequences similar to antisense oligonucleotides, but nevertheless are as catalytic as ribozymes and are designed to specifically cleave target nucleic acids.

  Ribozyme molecules designed to catalytically cleave transcripts of target mRNAs to prevent translation of mRNAs encoding c-MAFs in which the activity of c-MAF should be inhibited may also be used . Ribozymes are enzymatic RNA molecules capable of catalyzing the cleavage of specific RNAs (for a review, see Rossi, Current Biology 4: 469-471, 1994). The mechanism of action of the ribozyme involves specific hybridization of the ribozyme molecule sequence to complementary target RNA and subsequent endonucleolytic cleavage event. The composition of the ribozyme molecule preferably comprises one or more sequences complementary to the target mRNA and a known sequence responsible for cleavage of the mRNA or a functionally equivalent sequence (see, eg, US Pat. No. 5,093,246) about).

  In one embodiment, the subject has International Patent Application No. PCT / IB2013 / 001204 and US Patent Application No. 13 / 878,114 (triple negative breast cancer and ER + breast cancer), International Patent Application No. PCT / US2014 / 026154 (Renal cell carcinoma), International Patent Application No. PCT / US2014 / 028722 (breast cancer), International Patent Application No. PCT / US2013 / 044584 (lung cancer), US Patent Application No. 14 / 050,262 and International Patent Application No. PCT / IB2013 / 002866 (prostate cancer), International Patent Application No. PCT / US2014 / 059506 (HER2 + breast cancer), US Patent Application No. 14 / 213,670 and International Patent Application No. PCT / US2014 / 028569 (metastatic Cancer) (each in its entirety) ) Any c-MAF inhibitor, antisense oligonucleotide, siRNA, DNA enzyme, ribozyme, inhibitory antibody, inhibitory peptide, negative c-MAF dominant or other disclosed herein). It is treated with a c-MAF inhibitory molecule. In some embodiments, c-MAF inhibitors are used to treat or prevent bone degradation.

  Alternatively, combination treatment may be performed in which two or more of the agents mentioned above are combined to treat and / or prevent metastasis, or the agent may be used in other supplements (e.g. It can be combined with calcium or vitamin D) or hormone treatment.

  In another aspect, the invention is an in vitro method for predicting metastasis of said cancer in a subject suffering from cancer, using a binding member disclosed herein. And determining if the c-MAF gene in the sample of said subject is amplified relative to the reference gene copy number, wherein c-MAF gene as compared to said reference gene copy number Amplification relates to in vitro methods that show an increased risk of developing metastasis. In some embodiments, the amplification is in the region of the 16q23 locus.

  In another aspect, the invention relates to an in vitro method for predicting the clinical outcome of a patient suffering from cancer, which method comprises transposing the c-MAF gene in a sample of said subject And determining the translocation of the c-MAF gene indicates a poor clinical outcome.

  In another aspect, the invention relates to an in vitro method for predicting the clinical outcome of a patient suffering from cancer, which method comprises transposing the c-MAF gene in a sample of said subject And determining the translocation of the c-MAF gene indicates a poor clinical outcome.

  In some embodiments, the translocation gene is from the region of the 16q23 locus. In some embodiments, the translocation gene is from any portion of the approximately 79, 392, 959 bp to 79, 663, 806 bp (centromeric to telomeric) chromosomal region of about chromosome 16. In some embodiments, the translocation gene is from a genomic region of about 79, 392, 959 bp to 79, 663, 806 bp of approximately chromosome 16 but excluding DNA repetitive elements.

  In another aspect, the present invention relates to an in vitro method (hereinafter referred to as the seventh method of the present invention) for predicting the clinical outcome of a patient suffering from cancer, which method comprises Determining whether the c-MAF gene in the sample of the subject is amplified relative to the reference gene copy number, wherein amplification of the c-MAF gene as compared to the reference gene copy number Indicate poor clinical outcome.

  One embodiment comprises, in the first step, determining whether the c-MAF gene is amplified in a sample of the subject. The determination of the amplification of c-MAF is performed essentially as described above. In a preferred embodiment, the sample is a tumor tissue sample. In a preferred embodiment, amplification of the c-MAF gene is determined by determining amplification of locus 16q23 or 16q22-q24. In another preferred embodiment, amplification of the c-MAF gene is determined by using a c-MAF gene specific probe or an antibody as described herein. In a second step, this embodiment comprises the step of comparing the copy number to the copy number of the control sample or the reference sample, wherein the c-MAF copy number is the c-MAF copy number of the control sample and This is indicative of a poor clinical outcome when compared to a large number.

  In a preferred embodiment, when the c-MAF gene copy number is greater than the copy number possessed by the reference sample or the control sample, the c-MAF gene is amplified as compared to the reference gene copy number. In one example, the genomic copy number of the c-MAF gene is at least a 2-fold increase (ie, 6 copies), 3-fold increase (ie, 8 copies), 4-fold, 5 in the test sample as compared to the control sample. C-MAF gene if it is increased by 2, 6, 7, 8, 8, 9, 10, 15, 20, 25, 30, 40, 45, or 50 times Is said to be "amplified". In another example, the genomic copy number of c-MAF gene per cell is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, The c-MAF gene is said to be "amplified" if it is 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.

  In some embodiments, the antibody is an antibody of International Patent Application No. PCT / IB2013 / 001204 and US Patent Application No. 13 / 878,114 (triple negative breast cancer and ER + breast cancer), International Patent Application No. PCT / US2014 / 026154 (Renal cell carcinoma), International Patent Application No. PCT / US2014 / 028722 (breast cancer), International Patent Application No. PCT / US2013 / 044584 (lung cancer), US Patent Application No. 14 / 050,262 and International Patent Application No. PCT / IB2013 / 002866 (prostate cancer), International Patent Application No. PCT / US2014 / 059506 (HER2 + breast cancer), US Patent Application No. 14 / 213,670 and International Patent Application No. PCT / US2014 / 028569 (metastatic Cancer) (each, the whole As used in any of the methods described in to) incorporated herein by reference.

Methods of Treatment The binding members (eg, antibodies) of the invention may be administered in the form of a pharmaceutical composition, which may comprise at least one component in addition to the binding member. Thus, the pharmaceutical composition according to the invention, and the pharmaceutical composition for use according to the invention, in addition to the active ingredient, are pharmaceutically active excipients, carriers, buffers, stabilizers or well known to the person skilled in the art May contain other substances. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material depends on the route of administration, which may be oral, inhalation, intratracheal, topical, intravesical or by injection, as discussed below.

  Pharmaceutical compositions for oral administration may be in tablet, capsule, powder, liquid or semi-solid form. The tablets may comprise a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions generally comprise a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may also be included.

  Buffers such as phosphates, citrates and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl Alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3'-pentanol; and m-cresol); low molecular weight polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; Hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides Disaccharides, and other carbohydrates (including glucose, mannose or dextrin); chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt forming counterions such as sodium; metal complexes such as Zn protein complex); and / or preservatives including non-ionic surfactants such as TWEENTM, PLURONICSTM, or polyethylene glycol (PEG), stabilizers, buffers, antioxidants, and / or Other additives may be used as needed.

  Binding members (eg, antibodies) of the invention may be formulated in liquid, semi-solid or solid form, depending on the physicochemical properties of the molecule and the delivery route. The formulation may comprise an excipient or combination of excipients such as sugars, amino acids, and surfactants. Liquid formulations can include a wide range of antibody concentrations and pH. Solid formulations may be produced, for example, by lyophilisation, spray drying or drying by supercritical fluid technology. The formulation of the binding member depends on the desired delivery route. Binding members may be prepared with carriers that protect the binding member from rapid release, such as controlled release formulations including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are known to those skilled in the art (Robinson, (1978) Sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York).

  The compositions may be administered alone or in combination with other treatments, either simultaneously or sequentially depending on the condition to be treated.

  Binding members (e.g., antibodies) of the invention may be used as part of a combination therapy in conjunction with additional pharmaceutical ingredients. Combination treatment may be used to provide a significant synergistic effect, in particular the combination of a binding member of the invention and one or more other antibodies. The binding members of the invention may be administered simultaneously or sequentially, or with one or more additional therapeutic agents, for the treatment of one or more of the conditions listed herein. It can be administered as a combined preparation.

  The binding member (e.g., antibody) of the invention and one or more of the additional pharmaceutical ingredients described above may be used in the manufacture of a medicament. Because the medicament may be for separate or combined administration to an individual, it may comprise the binding member and additional components as a combined preparation or as separate preparations. Separate preparations may be used to facilitate separate sequential or simultaneous administration, and to allow administration of the components by different routes, such as oral and parenteral administration.

According to the invention, the provided compositions can be administered to a mammal. Administration is usually by a "therapeutically effective amount", which is sufficient to show benefit to the patient. Such benefit may be at least amelioration of at least one symptom. The actual amount administered and the rate and time-course of administration will depend on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the combination. It depends on the type of member, the mode of administration, the dosing schedule, as well as other factors known to the medical practitioner. Prescription of treatment (e.g. determination of dosage etc) is within the responsibility of general practitioners and other medical doctors and depends on the severity and / or progression of symptoms of the disease being treated It can depend. Appropriate doses of antibody are well known in the art (Ledermann et al. (1991) Int. J. Cancer 47: 659-664; Bagshawe et al. (1991) Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). The particular dosages indicated herein or as indicated in the Physician's Desk Reference (2009) may be used as appropriate for the type of medicament being administered. Therapeutically effective amounts or appropriate doses of binding members (eg, antibodies) of the invention can be determined by comparing their in vitro activity and in vivo activity in animal models. Methods for extrapolating from humans to effective doses in mice and other test animals are known. The exact dose is such that the antibody is for diagnosis, prevention or treatment, the size and location of the area to be treated, the exact nature of the antibody (eg whole antibody or fragment), and the antibody It depends on many factors, including the nature of any detectable label or other molecule. Typical antibody doses are in the range of at least about 100 μg to about 1 g for systemic application and at least about 1 μg to about 1 mg for topical application. More initial loading doses may be administered followed by one or more lower doses. Typically, the antibody is a whole antibody, such as an IgG ₁ isotype. This is a single treatment dose for adult patients, which may be proportionally adjusted for children, infants and neonates, and may be adjusted proportional to molecular weight for other antibody formats. The treatment may be repeated daily, twice weekly, weekly or monthly intervals at the discretion of the physician. The treatment may be every 2 to 4 weeks for subcutaneous administration and every 4 to 8 weeks for intravenous administration. The treatment may be periodic, and the period between administrations may be about 2 weeks or more, for example about 3 weeks or more, about 4 weeks or more, or about once a month . The treatment may be given before and / or after transplantation and / or may be directly administered or applied to the anatomic site of the surgical procedure.

Nucleic Acids The invention further provides an isolated nucleic acid encoding a binding member (eg, an antibody) of the invention. The nucleic acid may comprise DNA and / or RNA. In one embodiment, the invention provides a nucleic acid encoding an CDR or CDR set or _VH domain or _VL domain of the invention or an antigen binding site or antibody molecule of an antibody as defined above. In some embodiments, the polynucleotide has at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about about SEQ ID NO: 38, 40, and 42 It encodes a polypeptide comprising 100% identical heavy chain CDRs. In some embodiments, the polynucleotide is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about about SEQ ID NO: 26, 28, and 30 It encodes a polypeptide comprising 100% identical heavy chain CDRs. In some embodiments, the polynucleotide encoding V _H is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 99% with SEQ ID NO: 15. 100% identical. In some embodiments, the polynucleotide encoding V _H is SEQ ID NO: 15. In some embodiments, the polynucleotide encoding the heavy chain is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 99% with SEQ ID NO: 14. 100% identical. In some embodiments, the polynucleotide encoding a heavy chain is SEQ ID NO: 14. In some embodiments, the polynucleotide encoding V _L is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 99% with SEQ ID NO: 20 100% identical. In some embodiments, the polynucleotide encoding V _L is SEQ ID NO: 20. In some embodiments, the polynucleotide encoding the light chain is at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 99% with SEQ ID NO: 18 100% identical. In some embodiments, the polynucleotide encoding the light chain is SEQ ID NO: 18.

  In some embodiments, the invention is directed to a vector comprising a polynucleotide encoding any of the antigen binding members described herein. In some embodiments, the host cell comprises a vector. The invention also provides constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one of the above-mentioned polynucleotides.

In some embodiments, the present invention is directed to host cells comprising a polynucleotide encoding any of the antigen binding molecules described herein. The invention also provides a recombinant host cell comprising one or more of the above constructs. The nucleic acid encoding any CDR or set of CDRs or _VH domain or _VL domain or antigen binding site of an antibody or antibody molecule as such, as is a method of producing a coding product comprising expression from the encoding nucleic acid Form an aspect of the invention. Advantageously, expression may be achieved by cultivating said recombinant host cell containing the nucleic acid under appropriate conditions. Following production by expression of binding members comprising V _H or V _L domains disclosed herein, binding members are any suitable technique known in the art and considered suitable. It can be isolated and / or purified using.

  The nucleic acid according to the invention may comprise DNA or RNA and may be wholly or partially synthetic. References to nucleotide sequences set forth herein encompass, unless the context requires otherwise, DNA molecules having the specified sequence and RNA molecules having the specified sequence in which T is replaced with U. Do.

In some embodiments, the invention is a method of producing an antibody or fragment thereof that binds human c-MAF, as described herein, such that the nucleic acid is expressed and the antibody is produced. A method comprising the step of culturing any of the host cells. A still further aspect provides a method of producing a binding member comprising a _VH variable domain and / or a _VL variable domain of the present invention, comprising the step of expressing from the encoding nucleic acid. Such methods under conditions for production of V _H variable domain and / or V _L variable domain of the antibody may comprise the step of culturing a recombinant host cell.

  The method of production may comprise the steps of isolation and / or purification of the product. The method of production may comprise the step of formulating the product into a composition comprising at least one additional component such as a pharmaceutically active excipient.

  Systems for cloning and expression of polypeptides in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and insect cells as well as transgenic plants and animals. Expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. For a review, see, eg, Pluckthun, (1991) BioTechnology 9: 545-551. A common bacterial host is E. coli.

  Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for the production of binding members (Chadd & Chamow, (2001) Curr. Op. Biotech. 12: 188-194; Andersen & Krummen, (2002). Curr. Op. Biotech. 13: 117; Larrick & Thomas, (2001) Curr. Op. Biotech. 12: 411-418). Mammalian cell lines available in the art for expression of heterologous polypeptides include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, NS0 mouse melanoma cells, YB 2/0 rat These include myeloma cells, human fetal kidney (HEK) cells, human fetal retinal cells, and many others.

  If necessary, appropriate vectors can be selected or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes, and other sequences. The vector may optionally be a plasmid, phagemid or viral vector, such as a retroviral vector (Sambrook & Russell, Molecular Cloning: a Laboratory Manual: 3rd edition, 2001, Cold Spring Harbor Laboratory Press). For example, many known techniques and protocols for manipulation of nucleic acids in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells, and gene expression and analysis of proteins are described in Ausubel et al., Eds. Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, John Wiley & Sons, 4th edition 1999.

  A further aspect of the invention provides host cells containing the nucleic acids disclosed herein. Such host cells can be maintained in vitro and grown in tissue culture. Such host cells can also be maintained in vivo, for example, to produce binding members in ascites fluid. The presence of host cells in vivo may allow for intracellular expression of binding members of the invention as "intrabodies" or intracellular antibodies. Intrabodies can be used for gene therapy.

  Yet further embodiments provide a method comprising introducing a nucleic acid of the invention into a host cell. The introduction may use any available technology. In the case of eukaryotic cells, suitable techniques include calcium phosphate transfection, DEAE-dextran, electroporation, liposome-mediated transfection, and in the case of retrovirus or other viruses such as vaccinia virus or insect cells. May include transduction using baculovirus or any combination thereof. Introduction of nucleic acids into host cells, particularly eukaryotic cells, may use virus based systems or plasmid based systems. The plasmid system may be maintained episomally or may be integrated into the host cell genome or artificial chromosomes. Integration may be by random or targeted integration of one or more copies at a single locus or multiple loci. In the case of bacterial cells, suitable techniques may include calcium chloride transformation, electroporation, and transfection using bacteriophage.

  Following introduction, expression from the nucleic acid may be caused or allowed to occur, for example, by culturing host cells under conditions for expression of the binding member. Purification of the expression product can be achieved by methods known to those skilled in the art.

  The nucleic acids of the invention may be integrated into the genome (e.g. chromosome) of the host cell. Integration may be promoted by the inclusion of sequences which promote recombination with the genome, in accordance with standard techniques.

  The invention also provides a method comprising using the construct in an expression system to express the binding member or polypeptide.

  Thus, in some embodiments, binding members according to the present invention may be used in methods of diagnosis or treatment of disorders associated with increased c-MAF infection.

Kits of the Invention Also provided is a kit comprising a binding member (eg, an antibody) according to any aspect or embodiment of the invention. In another embodiment, the kit is for predicting metastasis of the cancer in a subject suffering from cancer, the kit comprising: a) c-MAF in a sample of the subject Means for quantifying the expression level; and b) means for comparing the expression level of quantified c-MAF in the sample with the reference c-MAF expression level. In some embodiments, the means used is optical density measurement or histopathological scoring to quantify c-MAF immunohistochemical staining, where-represents a non-positive tumor, +, ++, +++ represent different positive levels.

  In some embodiments, the immunohistochemical staining is a tissue microarray immunohistochemical staining. In some embodiments, the means is a reagent used to perform immunohistochemical staining. In some embodiments, the reagents are used to prepare formalin fixed and / or paraffin embedded (FFPE) cell samples or tissue samples for immunohistochemical staining. A description of immunohistochemistry can be found in US Pat. No. 8,785,150, which is incorporated herein by reference in its entirety.

  In some embodiments, the means is a reagent used to perform histopathological scoring. In some embodiments, the means for comparing the quantified c-MAF expression level to a reference c-MAF expression level comprises a quantifiable internal reference standard of c-MAF. In some embodiments, the metastasis is a bone metastasis. In some embodiments, the bone metastasis is osteolytic bone metastasis.

  In another aspect, the invention relates to a kit for predicting the clinical outcome of a subject suffering from bone metastasis from cancer, the kit comprising: a) c-MAF in a sample of said subject Means for quantifying the expression level; and b) means for comparing the expression level of quantified c-MAF in said sample with the reference c-MAF expression level.

  In another aspect, the invention relates to a kit for determining a treatment for a subject suffering from cancer, which kit comprises: a) quantifying the expression level of c-MAF in a sample of said subject B) means for comparing the expression level of quantified c-MAF in said sample with a reference c-MAF expression level; and c) based on comparing the quantified expression level with the reference expression level And means for determining a treatment for preventing and / or reducing bone metastasis in said subject.

  In another embodiment, the present invention is predetermined to correlate i) with a means for quantifying the expression level of c-MAF in a sample of the afflicted subject, and ii) the risk of bone metastasis The present invention relates to a kit comprising one or more c-MAF gene expression level indicators.

  In some embodiments, the present invention provides a kit for predicting bone metastasis of cancer in a subject suffering from cancer, the kit comprising: a) in a tumor sample of said subject A polypeptide encoded by an antigen binding molecule or fragment thereof described herein or a polynucleotide described herein, for use in quantifying the expression level of c-MAF of 2.) A means is provided for comparing the expression level of quantified c-MAF in the sample with the reference c-MAF expression level.

  The means for quantifying the expression level of c-MAF in a sample of said subject comprising amplification and translocation of the 16q23 and 16q22-24 loci have been described in detail above.

  In one embodiment, the means for quantifying expression comprises a set of antibodies. In some embodiments, the means for quantifying expression further comprises probes and / or primers that specifically bind to the c-MAF gene and / or specifically amplify the c-MAF gene.

  In certain embodiments, the cancer is breast cancer, lung cancer, prostate cancer, or kidney cancer.

  All of the specific embodiments of the methods of the invention are applicable to the kits of the invention and their use.

  In the kit, a binding member (eg, an antibody) can be labeled to allow one to determine its reactivity in a sample, for example as described further below. Furthermore, the binding member may or may not be bound to a solid support. In general, the components of the kit are sterile and in sealed vials or other containers. The kit can be used in diagnostic analysis or other methods where binding members are useful. The kit can include instructions for use of the components in a method (eg, a method according to the invention). Auxiliary materials to aid in such methods, or which make it possible to carry out such methods, can be included in the kits of the invention. As an adjunct material, a second different binding member that binds to the first binding member, the second different binding being conjugated to a detectable label (eg, a fluorescent label, a radioactive isotope or an enzyme) Members can be mentioned. Antibody based kits may also include beads for performing immunoprecipitation. Each component of the kit is generally in its own appropriate container. Thus, these kits generally comprise separate containers appropriate for each binding member. Additionally, the kit can include an assay and instructions for performing a method to interpret and analyze data obtained from performing the assay.

Example 1
Construction of a c-MAF-Specific Antibody The antibody INB-1-11-8 is raised against an epitope corresponding to amino acids 83-EQKAHLEDYYWMTGYPQ-100 (18a.a.) (SEQ ID NO: 22) of c-MAF of human origin. I did. The epitope was bound to KLH (NAc-EQKAHLEDYYWMTGYPQ-Ahx-C-KLH (20a.a.)). This antibody was compared to M153 (Santa Cruz Biotechnologies Inc.). The M153 antibody was raised against an epitope corresponding to amino acids 19-171 of c-MAF of mouse origin. c-MAF is highly conserved between human and mouse (see alignment in FIG. 2).

  The INB-1-11-8 light chain sequence (FIG. 8) is SEQ ID NO: 20 (leader (SEQ ID NO: 24); framework 1 (SEQ ID NO: 25); CDR1 (SEQ ID NO: 26); framework 2 (sequence CDR2 (SEQ ID NO: 28); Framework 3 (SEQ ID NO: 29); CDR3 (SEQ ID NO: 30); Junction (SEQ ID NO: 31); LC part (SEQ ID NO: 32)).

The human germinal sequence closest to the light chain of the INB-1-11-8 light chain sequence is as follows:

  The INB-1-11-8 heavy chain sequence (FIG. 8) is SEQ ID NO: 16 (leader (SEQ ID NO: 36); framework 1 (SEQ ID NO: 37); CDR1 (SEQ ID NO: 38); framework 2 (sequence CDR2 (SEQ ID NO: 40); Framework 3 (SEQ ID NO: 41); CDR3 (SEQ ID NO: 42); Junction (SEQ ID NO: 43) HC part (SEQ ID NO: 44)).

The closest human embryo sequence to the INB-1-11-8 heavy chain sequence is:

  Maf antibody sensitivity was calculated in the range of crescent dilution of primary antibody from 1:10 to 1: 1000. Antibody specificity was determined using parental and Maf overexpression (long and short isoforms of Maf) MCF7, T47D (The American Type Culture Collection; obtained from ATCC) and 0990 human breast cancer cells. The formalin fixed cell pellet was processed using standard immunohistochemistry procedures. Specificity was also shown in ectopic MCF7 and MCF7-Maf (long and short isoforms) xenografts in balb-c mice [mouse type]. Sections from the same specimen which were incubated with normal rabbit IgG2 (IS600, Dako) instead of primary antibody were used as negative control.

Antigen-specific ELISA
Antibody sensitivity to the antigen was tested using an antigen specific ELISA. For this purpose, the above epitope (peptide 1) was conjugated to BSA. This was bound to the plate surface on which the ELISA was performed and washed. Then, INB-1-11-8 c-MAF specific antibody was applied so that it could bind to the antigen. After incubation time and washing with TBST, the binding of the primary antibody to the antigen was scored using a secondary antibody specific for rabbit antibody, which was conjugated to alkaline phosphatase. Several dilutions were used to test the titer of the antibody, which is a measure of the affinity of the antibody for its antigen (FIG. 3A).

Western Blot c-MAF antibody specificity by Western Blot was calculated in the range of crescent dilution of primary antibody 1: 50 to 1: 250. Specificity using parent and Maf overexpression (long and short isoforms of Maf) MCF7, T47D, 293T (The American Type Culture Collection; obtained from ATCC) and MCF7-derived BoM2 human breast cancer cells with a tendency to metastasize bone It was determined. Cell pellets were processed by standard procedures as described (Tarragona et al., J. Biol. Chem. (2012) 287: 21346-55) and results were confirmed by Western blot from total lysates (Figure 3B).

c-MAF Immunostaining Immunostaining was performed using 3 μm tissue sections and placed on positively charged glass slides in a Dako Link platform. After deparaffinization, heat antigen retrieval was performed in 0.01 mol / L citrate based buffer solution (Dako) at pH 6.1. Quenched endogenous peroxidase. Mouse polyclonal anti-cMaf antibody was used at room temperature, 1: 100 dilution for 30 minutes, followed by incubation with peroxidase conjugated anti-rabbit Ig dextran polymer (Flex +, Dako). The sections were then visualized with 3,3′-diaminobenzidine (DAB) and counterstained with hematoxylin (FIG. 3C).

Example 2
Analysis of the interaction between monoclonal rabbit antibody and antigen c-MAF a) Analysis of cMaf protein preparation by SDS-PAGE In order to confirm the purity of the antigen preparation, using SDS-PAGE under reducing conditions , C-MAF (Q1) was compared to a commercial BSA standard. Three amounts (800 ng, 550 ng and 275 ng) of c-MAF (Q1) were compared to four amounts (750 ng, 500 ng, 250 ng and 125 ng) of BSA. FIG. 4 shows SDS-PAGE gel after Coomassie staining.

The SDS-PAGE gel shows two different bands of about 25 kDa (about 60%) and 20 kDa (*) (about 40%) for c-MAF (Q1). The calculated molecular weight of the molecule based on the sequence information is 19.2 kDa and such a difference between the calculated size and the apparent size in SDS-PAGE is common. As shown in the vial, for c-MAF (Q1), the nominal concentration appears to have a lower (less than one-third) concentration.
Mr = 19.2 kDa; pI5, 6; no consensus N-glycosylation site; two cysteines

b) Analysis of c-MAF protein preparation by densitometric analysis (CFCA) without calibration In order to determine the active concentration of c-MAF (Q1) binding to INB-1-11-8, the CFCA method was used. This method relies on the change in binding rate with various flow rates when transport of molecules to the sensor surface is limited by diffusion. The concentration is calculated from the measured binding rate and the molecular weight of the analyte and the estimated diffusion coefficient. A high density of binding at the ligand surface was achieved by capturing> 3000 RU of INB-1-11-8. Binding rates were measured at flow rates of 5, 20 and 100 μL / min. FIG. 5 shows an example of a sensorgram of CFCA analysis. A concentration of 1.3 mg / ml was determined for c-MAF (Q1). These results support the observations made in SDS-PAGE analysis at estimated concentrations compared to BSA standards. Assuming that both the 25 kDa band observed by SDS-PAGE and the smaller 20 kDa band have antibody binding activity, CFCA measured the concentration determined for c-MAF (Q1). In this case, the amounts of bands observed on the SDS-PAGE gel were summed to estimate the active concentration of the antigen preparation.

c) Kinetic Analysis In order to determine the affinity constant between antibody INB-1-11-8 and c-MAF (Q1) preparation, kinetic measurements were performed under the following conditions:

Experimental conditions and equipment: Biacore T200
Running buffer: HBS-EP, 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4
Assay temperature: 25 ° C
Sensor surface: recombinant protein A; immobilized by standard amine coupling (EDC / NHS chemistry)

  Test measurements were performed to confirm protein A binding of antibody INB-1-11-8 and to select appropriate capture levels for analyte kinetic measurements. When a capture level of 240 RU was selected for measurement with c-MAF (Q1), the predicted Rmax of the two 19.6 kDa antigens bound per antibody molecule resulted to about 64 RU. Selecting a higher capture level of 980 kDa to generate a sufficiently high binding signal for the peptide resulted in an expected Rmax of about 30 RU. Serial dilutions of antigen were injected for 180 seconds followed by 600 seconds of buffer for determination of the off rate (see Figure 6). For the c-MAF fragment, the molar concentration used for serial dilution was calculated based on the concentration determined by CFCA. Kinetic measurements were performed twice to confirm the binding behavior. The figure shows fast binding of the analyte at protein concentrations above 10 nM.

In order to evaluate the affinity of the interaction between c-MAF (Q1) and antibody INB-1-11-8 (Table 1), assuming that Rmax is 23 RU, based on a 1: 1 interaction model A single concentration fit was performed (Figure 7). Based on this preliminary data, affinities in the nM range were roughly estimated for the interaction. Binding is characterized by a very fast association rate and a fast dissociation rate (ie, the binding is not very tight).

  c-MAF (Q1): Fragment of c-MAF used (including Aa19-208)

reagent:
C-MAF specific monoclonal antibody (rabbit) INB-1-11-8 (clone ID 11-8; Lot: 11-8); concentration 1.7 mg / ml; 3 aliquots, about 200 μL
C-MAF (Q1) fragment HBS-EP (running buffer)
・ CM5 sensor chip ・ Amine coupling kit ・ Protein-A
・ 30 mM HCl (regeneration buffer)

Example 3
Validation Breast Cancer Primary Tumor Sample Cohorts The ability of the antibodies to identify and predict bone metastases was tested in a human breast tumor cohort. The validation set consisted of over 380 primary breast cancer specimens from patients with stage I, II or III breast cancer and annotated clinical follow-up (Rojo F., Ann Oncol 23 (5): 1156) -1164 (2012)). Tissue microarrays were processed according to standard procedures. Tumors were classified according to standard clinicopathological parameters and then appropriate statistical analysis was performed to confirm that c-MAF (MAF) protein expression in these tumors correlates with bone metastasis events .

Statistical analysis in this second cohort was based on the following assumptions:
i) Diagnostic ability

Diagnosability was assessed by comparing the AUG of the ROC curve. The sensitivity (Se), specificity (Sp), positive predictive value (PPV) and negative predictive value (NPV) were calculated for each of the classification categories based on most predictors (MAF IHC levels) (FIG. 9). Cutoffs for selecting MAF positive and negative tumors were established based on received operating curve parameters (ROC).
ii) Comparison of baseline characteristics (Figure 10).

The Kruskal-Wallis test was used to test for differences in mean age. Where applicable, chi-square tests were used to compare categorical variables.
iii) Prognostic role-Cox regression modeling of outcome (time to bone metastasis) and hazard ratio were calculated (Figure 11).

  The examples and embodiments described herein are for illustrative purposes only, and it is understood that various modifications or alterations in consideration thereof are suggested to those skilled in the art and are included in the spirit and scope of the present application. Be done.

  All publications, patents, patent applications, internet sites and accession numbers / database sequences (including both polynucleotide sequences and polypeptide sequences) cited herein have their respective individual publications, patents, The patent application, internet site or accession number / database sequence is incorporated herein by reference in its entirety for all purposes, to the same extent as if specifically and individually indicated to be incorporated by reference. Incorporated by reference.

Claims (48)

  A binding member or a functional fragment or variant thereof that specifically binds to the epitope encoded by SEQ ID NO: 22.   A heavy chain CDR1 specifically binding to human c-MAF and comprising the amino acid sequence of SEQ ID NO: 38, and / or a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and / or a heavy chain comprising the amino acid sequence of SEQ ID NO: 42 A light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 26, and / or a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and / or a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 30; A binding member according to claim 1 or a functional fragment or variant thereof.   3. The binding member or functional fragment or variant thereof according to claim 1 or 2, wherein said binding member is an antibody.   4. The binding member or functional fragment or variant according to claim 3, wherein said antibody is a rabbit antibody, a chimeric antibody or a humanized antibody. 5. The binding member or functional fragment or variant thereof according to claim 4, comprising a _VH domain having a sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 17. 6. The binding member or functional fragment or variant thereof according to claim 5, comprising a V _H domain having a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 17. 7. The binding member or functional fragment or variant thereof according to claim 6, comprising a V _H domain having a sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 17. 8. The binding member or functional fragment or variant thereof according to claim 7, comprising a V _H domain having a sequence comprising the amino acid sequence of SEQ ID NO: 17. 9. The binding member or functional fragment or variant thereof according to any one of claims 1 to 8, comprising a V _L domain having a sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 21. 10. The binding member or functional fragment or variant thereof according to claim 9, comprising a V _L domain having a sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 21. 11. The binding member or functional fragment or variant thereof according to claim 10 comprising a V _L domain having a sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 21. 12. The binding member or functional fragment or variant thereof according to claim 11, comprising a V _L domain having a sequence comprising the amino acid sequence of SEQ ID NO: 21.   13. The binding member or functional fragment or variant thereof according to any one of claims 1 to 12, comprising a heavy chain sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 16.   14. The binding member or functional fragment or variant thereof according to claim 13, comprising a heavy chain sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 16.   15. The binding member or functional fragment or variant thereof according to claim 14, comprising a heavy chain sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 16.   16. The binding member or functional fragment or variant thereof according to claim 15, comprising a heavy chain sequence comprising the amino acid sequence of SEQ ID NO: 16.   17. The binding member or functional fragment or variant thereof according to any one of claims 1 to 16, comprising a light chain sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 20.   18. The binding member or functional fragment or variant thereof according to claim 17, comprising a light chain sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 20.   19. The binding member or functional fragment or variant thereof according to claim 18, comprising a light chain sequence at least 95% identical to the amino acid sequence of SEQ ID NO: 20.   20. The binding member or functional fragment or variant thereof according to claim 19, wherein said antibody or fragment thereof comprises a light chain sequence comprising the amino acid sequence of SEQ ID NO: 20.   A polynucleotide encoding the binding member according to any one of claims 1 to 20 or a functional fragment or variant thereof.   22. The polynucleotide of claim 21, wherein the polypeptide encodes an antigen binding molecule or a fragment thereof.   The polynucleotide according to claim 21 or 22, wherein said binding member or functional fragment or variant thereof is an antibody. 24. The polynucleotide of any one of claims 21-23, comprising a V _H domain having a sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 15. 25. The polynucleotide of claim 24, which encodes a V _H domain having a sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 15. 26. The polynucleotide of claim 25 which encodes a V _H domain having a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 15. 27. The polynucleotide of claim 26, which encodes a V _H domain having a sequence identical to the nucleotide sequence of SEQ ID NO: 15. 28. The polynucleotide of any one of claims 21-27, which encodes a V _L domain having a sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 20. 29. The polynucleotide of claim 28, which encodes a V _L domain having a sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 20. 30. The polynucleotide of claim 29, which encodes a V _L domain having a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 20. 31. The polynucleotide of claim 30, which encodes a V _L domain having a sequence identical to the nucleotide sequence of SEQ ID NO: 20.   32. The polynucleotide of any one of claims 21 to 31 encoding a heavy chain having a sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 14.   33. The polynucleotide of claim 32, which encodes a heavy chain having a sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 14.   34. The polynucleotide of claim 33, which encodes a heavy chain having a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 14.   35. The polynucleotide of claim 34 which encodes a heavy chain having a sequence identical to the nucleotide sequence of SEQ ID NO: 14.   36. The polynucleotide of any one of claims 21-35, which encodes a light chain having a sequence at least 80% identical to the nucleotide sequence of SEQ ID NO: 18.   37. The polynucleotide of claim 36, which encodes a light chain having a sequence at least 90% identical to the nucleotide sequence of SEQ ID NO: 18.   38. The polynucleotide of claim 37, which encodes a light chain having a sequence at least 95% identical to the nucleotide sequence of SEQ ID NO: 18.   39. The polynucleotide of claim 38, which encodes a light chain having a sequence identical to the nucleotide sequence of SEQ ID NO: 18.   40. The polynucleotide of claims 21-39, wherein the amino acid encoded by the polynucleotide binds to the epitope set forth in SEQ ID NO: 22.   21. The binding member or functional fragment or variant thereof according to any one of claims 1 to 20 which binds human c-MAF with an affinity (KD) of at least about 1.5 nM or less.   42. A binding member or functional fragment or variant thereof according to claim 41 which binds human c-MAF with an affinity (KD) of at least about 1.2 nM or less.   43. The binding member or functional fragment or variant thereof according to claim 42, which binds human c-MAF with an affinity (KD) of at least about 1.1 nM or less.   41. A vector comprising the polynucleotide of any one of claims 21-40.   The polynucleotide according to any one of claims 21 to 40, the vector according to claim 44, or the binding member according to any one of claims 1 to 19 or 41 to 43 , Host cells.   46. A method of producing an antigen binding member comprising culturing the host cell of claim 45.   47. A method of detecting c-MAF using the host cell of claim 45 or an antigen binding member produced by the method of claim 46. A binding member or a functional fragment or variant thereof that competes with INB-1-11-8 for binding to the epitope encoded by SEQ ID NO: 22.